Method of determining drug toxicity

Abstract

The present disclosure relates to the field of methods for the determination of drug toxicity, and more particularly cardiotoxicity associated with an anti-cancer agent. Further, the present disclosure provides related methods of treatment and stratifying patients with cancer for a clinical trial utilising such agents.

Description

[0001] Method of determining drug toxicity

[0002] Cross-reference to related applications

[0003] The present application claims priority from Australian Provisional Patent Application No. 2024902481 filed on 9 August 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] Technical field

[0005] The present disclosure relates to the field of methods for the determination of drug toxicity, and more particularly cardiotoxicity associated with an anti-cancer agent. Further, the present disclosure provides related methods of treatment and stratifying patients with cancer for a clinical trial utilising such agents.

[0006] Background

[0007] Cardiotoxicity remains a common side effect that may result from the administration of a broad range of clinically available anti -cancer drug therapies or treatments to patients. Moreover, and despite the diverse range of such therapies now available to patients, the adequate and appropriate treatment of cancer remains elusive for many individuals. Thus, the treatment of cancer in patients with minimal or no side effects, such as cardiotoxicity, is an area of ongoing clinical importance. Accordingly, there remains an unmet clinical need for methods that can effectively and accurately predict therapeutic efficacy and the risk of cardiotoxicity for drug therapies, particularly anti-cancer treatments, in patients in need thereof.

[0008] Summary

[0009] The present disclosure is based on the utilisation of cardiac cells produced from patient- derived blood cells, and more particularly PBMCs, together with patient-specific efficacy data, such as that generated in vitro from patient-derived cancer cells, may be capable of accurately predicting therapeutic efficacy and potential cardiotoxicity of anti-cancer agents in a patientspecific manner. By extension, such methods demonstrate promise in classifying cancer patients according to the risk of treatment failure and / or cardiotoxicity. These methods may also aid in predicting what cardioprotective agents may be utilized for particular patients based on the nature and degree of cardiotoxicity of the anti -cancer agents demonstrated by the present model. In a first aspect, the present disclosure provides a method of predicting the therapeutic efficacy and cardiotoxicity of an anti -cancer agent in a subject having cancer, said method including the steps of

[0010] (a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject; and

[0011] (b) determining a level of therapeutic efficacy for the anti -cancer agent in the subject’s cancer.

[0012] Suitably, based on the determinations made in steps (a) and (b), the method further including the step of initiating, continuing, modifying or discontinuing administration of the anticancer agent to the subject. For certain examples, the present method further includes the step of administering to the subject a therapeutically effective amount of the anti -cancer agent when the level of cardiotoxicity indicates or correlates with a low likelihood of cardiotoxicity in the subject and the level of therapeutic efficacy indicates or correlates with a high likelihood of therapeutic efficacy of the anti-cancer agent in the subject.

[0013] In a second aspect, the present disclosure provides a method of treating a cancer in a subject, said method including the steps of

[0014] (a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject;

[0015] (b) determining a level of therapeutic efficacy for the anti -cancer agent in the subject’s cancer; and

[0016] (c) initiating, continuing, modifying or discontinuing administration of the anti-cancer agent to the subject based on the determinations made in steps (a) and (b).

[0017] Referring to the above aspects, the blood-derived cells are suitably PBMCs.

[0018] The methods of the first and second aspects may further include the initial or earlier step of isolating the blood-derived cells from the subject.

[0019] The methods of the first and second aspects may further include the step of generating the cardiac cells from the blood-derived cells.

[0020] For the above aspects, the cardiac cells are suitably cultured, at least in part, in a 3- dimensional culture system, such as a cardiac organoid or a cardiac spheroid. In some examples, the cardiac organoid or the cardiac spheroid comprise cardiomyocytes, interstitial cells and vascular cells.

[0021] Suitably, the cardiac cells of the above aspects are cultured in a media comprising blood, plasma or serum derived or obtained from the subject. The methods of the first and second aspects may further include the step of administering to the subject a therapeutically effective amount of a cardioprotective agent in combination with the anti-cancer agent.

[0022] The methods of the first and second aspects may further include the step of culturing the cardiac cells in the presence of the cardioprotective agent and the anti-cancer agent. For such examples, the present methods may further include the step of determining whether contacting the cardiac cells with the cardioprotective agent in combination with the anti-cancer agent modulates the level of cardiotoxicity for the anti -cancer agent in the subject. Suitably, the cardioprotective agent is administered in combination with the anti-cancer agent when the level of cardiotoxicity indicates or correlates with a medium or high likelihood of cardiotoxicity in the subject and the level of therapeutic efficacy indicates or correlates with an intermediate or high likelihood of therapeutic efficacy of the anti-cancer agent in the subject. Moreover, the cardioprotective agent can be administered in combination with the anti-cancer agent when contacting the cardiac cells with the cardioprotective agent in combination with the anti-cancer agent reduces the level of cardiotoxicity for the anti -cancer agent in the cardiac cells.

[0023] Referring to the first and second aspects, determining the level of cardiotoxicity suitably comprises contacting the cardiac cells with a dose range or a plurality of doses or concentrations of the anti-cancer agent.

[0024] Suitably for the above aspects, the level of cardiotoxicity is determined at least partly by detecting or determining a level or presence of one or more of vascular damage, vascular dysfunction, cardiac cell viability, cardiac cell atrophy, cardiac cell necrosis, cardiac cell apoptosis, cardiac fibrosis, contractile dysfunction, electrophysiological dysfunction, mitochondrial damage, oxidative stress and inflammation.

[0025] Referring to the aforementioned methods, the anti-cancer agent may be selected from the group consisting of: a chemotherapeutic agent, a molecularly targeted agent, a T cell expressing a chimeric antigen receptor, an antibody or antigen-binding fragment thereof, an antibody-drug conjugate, an angiogenesis inhibitor, an immunotherapeutic agent, radiotherapy and any combination thereof.

[0026] Step (b) of the aforementioned aspects suitably includes determining the level of therapeutic efficacy for the anti -cancer agent in cancer cells obtained or derived from the subject’s cancer. In some examples, the cancer cells comprise a 2D culture, a 2D co-culture, a 2.5D culture, a 3D culture or a patient-derived xenograft. More particularly, the cancer cells suitably are or comprise a cancer cell line or a patient-derived xenograft. In this regard, the present methods may further include the initial or earlier step of isolating the cancer cells from the subject. Suitably, determining the level of therapeutic efficacy comprises contacting the cancer cells with a dose range (e.g., a plurality of doses or concentrations) of the anti-cancer agent.

[0027] In a third aspect, the present disclosure provides a method of stratifying a subject having cancer for a clinical trial of an anti -cancer agent, said method including the steps of:

[0028] (a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject;

[0029] (b) determining a level of therapeutic efficacy for the anti -cancer agent in the subject’s cancer; and

[0030] (c) stratifying the subject for the clinical trial based on the results of determining steps (a) and (b).

[0031] In a fourth aspect, the present disclosure provides a method of determining inclusion of a subject having cancer in a clinical trial of an anti-cancer agent, said method including the steps of:

[0032] (a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject;

[0033] (b) determining a level of therapeutic efficacy for the anti -cancer agent in the subject’s cancer; and

[0034] (c) determining whether the subject having cancer is included in the clinical trial based on determining steps (a) and (b).

[0035] Suitably, the aforementioned methods further include the step of monitoring the subject for cardiotoxicity during administration of the anti-cancer agent when the level of cardiotoxicity indicates or correlates with a medium or high likelihood of cardiotoxicity in the subject. In some examples, the monitoring step includes detecting a level or presence of one or more of vascular damage, vascular dysfunction, cardiac cell viability, cardiac cell atrophy, cardiac cell necrosis, cardiac cell apoptosis, cardiac fibrosis, contractile dysfunction, calcium signalling and action potential dysfunction, mitochondrial damage, oxidative stress and inflammation in the subject.

[0036] Suitably, the aforementioned methods further include the step of determining whether contacting the cardiac cells with a cardioprotective agent in combination with the anti-cancer agent reduces the level of cardiotoxicity for the anti-cancer agent in the subject. More particularly, the aforementioned methods further include the steps of: contacting the cardiac cells with a cardioprotective agent in combination with the anti-cancer agent; and determining whether the cardioprotective agent in combination with the anti-cancer agent reduces the level of cardiotoxicity for the anti-cancer agent in the subject.

[0037] Suitably, the aforementioned methods further include the step of determining a further level of cardiotoxicity for the anti-cancer agent in the cardiac cells at a time point following administration of the anti-cancer agent, wherein the cardiac cells are cultured in a media comprising blood, plasma or serum derived or obtained from the subject at or around the time point.

[0038] Brief description of the drawings

[0039] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0040] Figure 1: Schematic illustration of isolating patient-derived peripheral blood mononuclear cells (PBMCs) for cardiac spheroid (CS) formation. 1) Isolation of PBMCs from patient’s blood. 2) Growing and expanding PBMCs to erythroid progenitor cells (EPCs). 3) Reprogramming EPCs to induced pluripotent stem cells (IPSCs) using episomal vector. 4) Expanding IPSCs. 5) Differentiating IPSCs to IPSC-derived cardiomyocytes and IPSC-derived endothelial cells while expanding cardiac fibroblast. 6) Co-culturing the three cell types for cardiac spheroid formation.

[0041] Figure 2: DOX-mediated cardiotoxic effects differ in patient-derived cardiac spheroids. (A- G) Statistical analyses (n > 3) of DOX-mediated cardiotoxic effects in cardiac spheroids from patients Pl, P2, P3, P4, P5, P6 and P7 treated with 0 (CTL), 1, 5, 10, 20, and 40 pM doxorubicin. * p < 0.05 relative to control; Data are presented as mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparisons test.

[0042] Detailed description

[0043] General Techniques and Definitions

[0044] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in tissue engineering, stem cell technologies, genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0045] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology. Such procedures are described, for example in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, Second Edition., 1995), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984) and Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series.

[0046] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0047] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0048] Each feature of any particular aspect or embodiment or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment or embodiment of the present disclosure.

[0049] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0050] As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise. For example, a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an allergen” is a reference to one or more allergens.

[0051] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0052] Throughout this specification, the word “comprise’ or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0053] Throughout the present specification, various aspects and components of the disclosure can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 5, 5. 5 and 6, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

[0054] The term “about” in relation to a numerical value x is optional and means, for example, any number within 1, 5 or 10% of the referenced number. In certain examples, the term “about” encompasses the exact number recited.

[0055] The term “substantially” does not exclude “completely” (e.g., a composition which is “substantially free” from Y may be completely free from Y).

[0056] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

[0057] For the present disclosure, the database accession number or unique identifier provided herein for a gene or protein, as well as the gene and / or protein sequence or sequences associated therewith, are incorporated by reference herein.

[0058] Predicting the therapeutic efficacy and cardiotoxicity of anti-cancer agents

[0059] The utilisation of cardiotoxicity data generated from patient-specific cardiac cells derived from blood-derived cells, and more particularly peripheral blood mononuclear cells (PBMCs), together with patient-specific efficacy data or information in relation to an anti -cancer agent has the potential to accurately predict the clinical outcome of such anti-cancer agents in a patientspecific manner. Advantageously, such a method may allow a physician to determine both the therapeutic responsiveness and risk of cardiotoxicity for a particular anti -cancer agent in a subject and in doing so make appropriate, informed, and timely treatment decisions based on this information.

[0060] In a particular broad form, the present disclosure provides a method of predicting the cardiotoxicity of an anti-cancer agent in a subject having cancer, said method including the step of determining a level of cardiotoxicity for the anti-cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject.

[0061] Accordingly, in one form there is provided herein a method of predicting the therapeutic efficacy and cardiotoxicity of an anti -cancer agent in a subject having cancer, said method including the steps of:

[0062] (a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject; and

[0063] (b) determining a level of therapeutic efficacy for the anti-cancer agent in the subject’s cancer.

[0064] With respect to the aspects described herein, the term “subject” refers to any animal, for example, a mammalian animal, including, but not limited to humans, non-human primates, livestock (e.g., sheep, horses, cattle, pigs, donkeys), companion animals (e.g., pets such as dogs and cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs), performance animals (e.g., racehorses, camels, greyhounds) or captive wild animals. In various examples, the “subject” is a human, such as a male human or a female human. Typically, the terms “subject” and “patient” are used interchangeably herein, particularly in reference to a human subject.

[0065] As generally used herein, the terms “cancer”, “tumour”, “malignant” and “malignancy” refer to diseases or conditions, or to cells or tissues associated with the diseases or conditions, characterized by aberrant or abnormal cell proliferation, differentiation and / or migration often accompanied by an aberrant or abnormal molecular phenotype that includes one or more genetic mutations or other genetic changes associated with oncogenesis, expression of tumour markers, loss of tumour suppressor expression or activity and / or aberrant or abnormal cell surface marker expression. The term is also intended to encompass primary and secondary (or metastatic) cancers, as are known in the art.

[0066] Cancers may include any aggressive or potentially aggressive cancers, tumours or other malignancies known in the art, such as listed in the NCI Cancer Index at https: / / www.cancer.gov / types, including all major cancer forms such as sarcomas, carcinomas, lymphomas, leukaemias and blastomas, although without limitation thereto. These may include breast cancer, lung cancer inclusive of lung adenocarcinoma, cancers of the reproductive system inclusive of ovarian cancer, cervical cancer, uterine cancer and prostate cancer, cancers of the brain and nervous system, head and neck cancers, gastrointestinal cancers inclusive of colon cancer, colorectal cancer and gastric cancer, liver cancer, kidney cancer, skin cancers such as melanoma and skin carcinomas, blood cell cancers inclusive of lymphoid cancers and myelomonocytic cancers, cancers of the endocrine system such as pancreatic cancer and pituitary cancers, musculoskeletal cancers inclusive of bone and soft tissue cancers, although without limitation thereto. Exemplary cancers include those of the brain, head and neck, prostate, ovary, breast, cervix, lung and oesophagus, as well as melanoma, rhabdomyosarcoma, mesothelioma, squamous carcinomas of the skin and glioblastoma.

[0067] It is further envisaged that the methods described herein may include the initial or earlier step of diagnosing the cancer in the subject. Any diagnostic test or assay known in the art, including imaging modalities, biopsy and in vitro assays, such as those based on a biological sample derived from the subject in question, may be utilised in this regard.

[0068] The terms “predicting”, “predicting the likelihood of’ and “predicting the risk of’ as used herein refer to methods by which the skilled artisan can predict or estimate the therapeutic efficacy and / or cardiotoxicity of an anti -cancer agent in an individual having cancer. The term “predicting” does not refer to the ability to predict the outcome with 100% accuracy. Instead, the skilled artisan will understand that the term “predicting” refers to a forecast or estimate of an increased or a decreased probability, possibility or likelihood that a certain outcome will occur; that is, that an outcome (e.g., cardiotoxicity and / or therapeutic efficacy) is more or less likely to occur in an individual based on the assays, information or tests described herein.

[0069] The terms “determining”, “measuring”, “evaluating”, “assessing”, “calculating” and “assaying” are used interchangeably herein and may include any form of measurement known in the art, such as those described hereinafter. Such determining may include detecting the presence or absence of cardiotoxicity and / or therapeutic efficacy in the respective cells, tissues or organs obtained or derived from the subject. Such determining can also or alternatively include determining a level of cardiotoxicity and / or a level of therapeutic efficacy of an anti-cancer agent described herein.

[0070] As will be understood by the skilled person, the level of cardiotoxicity and / or the level of therapeutic efficacy of an anti -cancer agent described herein may be relatively (i) higher, increased or greater; or (ii) lower, decreased or reduced when compared to that of a reference or control population or a reference or threshold level thereof. For instance, a threshold level may represent the average or median level of cardiotoxicity or therapeutic efficacy in a group of control subjects, plus or minus 1, 2, 3 or more standard deviations. In various examples, a level, such as that of cardiotoxicity or therapeutic efficacy, may be classified as higher, increased or greater if it exceeds a mean and / or median level thereof observed in a reference or control population, such as a reference or control population treated with the anti -cancer agent in question, a placebo or no anticancer agent. In some examples, a level, such as that of cardiotoxicity or therapeutic efficacy, may be classified as lower, decreased or reduced if it is less than the mean and / or median expression level observed in a reference or control population, again such as a reference or control population treated with the anti-cancer agent in question, a placebo or no anti-cancer agent. In this regard, a reference or control population may be a group of subjects who have the same cancer type, subgroup, stage and / or grade as said subject for which the level of cardiotoxicity and / or the level of therapeutic efficacy of an anti-cancer agent are determined.

[0071] Terms such as “higher”, “increased” and “greater” as used herein refer to an elevated level of cardiotoxicity or therapeutic efficacy, when compared to a control, threshold or reference level thereof. The level of cardiotoxicity or therapeutic efficacy may be relative or absolute (i.e., relatively or absolutely higher, increased or greater). In some examples, the level of cardiotoxicity or therapeutic efficacy is higher, increased or greater if its level is more than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400% or at least about 500% above the level of a control, threshold or reference level thereof, such as that in the absence of the anti-cancer agent or a mean or median level thereof observed in a reference population.

[0072] The terms, “lower”, “reduced” and “decreased”, as used herein refer to a lower level of cardiotoxicity or therapeutic efficacy, when compared to a control, threshold or reference level thereof. The level of cardiotoxicity or therapeutic efficacy may be relative or absolute (i.e., relatively or absolutely lower, reduced or decreased). In some examples, the level of cardiotoxicity or therapeutic efficacy is lower, reduced or decreased if its level is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or 0.0001% of the level of a control, threshold or reference level thereof, such as that in the absence of the anti-cancer agent or a mean or median level thereof observed in a reference population.

[0073] Suitably, based on the determinations made in steps (a) and (b), the present method further includes the step of initiating, continuing, modifying or discontinuing administration of the anticancer agent to the subject.

[0074] By way of example, the present method may include the step of initiating or continuing administration of a therapeutically effective amount of the anti-cancer agent to the subject. For such examples, the level of cardiotoxicity suitably indicates or correlates with a low likelihood of cardiotoxicity for the anti -cancer agent in the subject. Alternatively, or additionally, for such examples, the level of therapeutic efficacy suitably indicates or correlates with an intermediate or high likelihood of therapeutic efficacy (i.e., drug responsiveness of the subject’s cancer) of the anti-cancer agent in the subject. In an alternative form, the present method can include the step of modifying or discontinuing administration of a therapeutically effective amount of the anti -cancer agent to the subject. In this regard, the subject may already be receiving the anti -cancer agent in question. For such examples, the level of cardiotoxicity suitably indicates or correlates with an intermediate or high likelihood of cardiotoxicity for the anti-cancer agent in the subject. By way of example, the subject may be administered a therapeutically effective amount of a cardioprotective agent in combination with the anti-cancer agent, as described in more detail herein. For such examples, the level of therapeutic efficacy suitably indicates or correlates with an intermediate or high likelihood of therapeutic efficacy of the anti-cancer agent in the subject. In other examples, the subject may be administered a lower or low dose of the anti-cancer agent, as described in more detail herein. Again, for such examples, the level of therapeutic efficacy suitably indicates or correlates with an intermediate or high likelihood of therapeutic efficacy of the anti-cancer agent, including the low dose thereof, in the subject. In further examples, administration of the anti-cancer agent to the subject is modified, stopped or discontinued based on one or both of the level of cardiotoxicity and the level of therapeutic efficacy determined for the anti -cancer agent. For such examples, the level of therapeutic efficacy suitably indicates or correlates with a low or intermediate likelihood, and more particularly a low likelihood, of therapeutic efficacy of the anti -cancer agent in the subject and / or the level of cardiotoxicity suitably indicates or correlates with an intermediate or high likelihood of cardiotoxicity for the anti-cancer agent in the subject.

[0075] Accordingly, the present method may further include the step of administering to the subject a therapeutically effective amount of the anti-cancer agent. This may be particularly when one or both of the level of cardiotoxicity indicates or correlates with a low likelihood of cardiotoxicity in the subject and the level of therapeutic efficacy indicates or correlates with an intermediate or high likelihood of therapeutic efficacy of the anti-cancer agent in the subject.

[0076] In view of the foregoing, the present method suitably further includes the step of administering to the subject a therapeutically effective amount of a cardioprotective agent in combination with the anti -cancer agent, as described in more detail herein.

[0077] The term “therapeutically effective amount” describes a quantity of a specified agent, such as an anti-cancer agent, a cardioprotective agent or a composition described herein, sufficient to achieve a desired effect in a subject being treated with that agent or composition. For example, this can be the amount of an anti-cancer agent and optionally one or more further therapeutic agents, necessary to treat, reduce, alleviate and / or prevent a cancer or a cancer-associated disease, disorder or condition. Suitably, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of a cancer. More particularly, a “therapeutically effective amount” may be an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease or prevent disease progression, such as cancer growth and / or metastasis.

[0078] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent useful for reducing, alleviating and / or preventing the diseases, disorders and conditions described herein will be dependent on the subject being treated, the type and severity of any associated disease, disorder and / or condition (e.g., disease progression), and the manner of administration of the therapeutic composition. As described in more detail below, a “therapeutically effective amount” also includes a low or reduced dose and a high or increased dose of an anti-cancer agent or treatment when compared to a standard therapeutic dose thereof.

[0079] Treating cancer

[0080] In view of the foregoing, further aspects of the present disclosure provide methods of treating a cancer in a subject.

[0081] Accordingly, in one form the present disclosure provides a method of treating a cancer in a subject, said method including the steps of:

[0082] (a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject;

[0083] (b) determining a level of therapeutic efficacy for the anti -cancer agent in the subject’s cancer; and

[0084] (c) initiating, continuing, modifying or discontinuing administration of the anti-cancer agent to the subject based on the determinations made in steps (a) and (b).

[0085] Suitably, the present method (and also other methods described herein) further includes the step of administering a cardioprotective agent to the subject. To this end, such a method may include the step of (d) initiating, continuing, modifying or discontinuing administration of a cardioprotective agent to the subject based on the determinations made in steps (a) and (b). For such examples, the level of cardiotoxicity and optionally the level of therapeutic efficacy in steps (a) and (b) respectively may be assessed in the presence of a cardioprotective agent. In this regard, the methods described herein may further include the steps of: contacting the cardiac cells with a cardioprotective agent in combination with the anti-cancer agent; and determining whether the cardioprotective agent in combination with the anti-cancer agent reduces the level of cardiotoxicity for the anti-cancer agent in the subject.

[0086] To this end, the present method may include the further step of determining a further level of cardiotoxicity for the anti -cancer agent and the cardioprotective agent in the cardiac cells of the subject. In this regard, the cardiac cells can be contacted or cultured simultaneously or sequentially (i.e., culturing with the anti-cancer agent and then the cardioprotective agent or vice versa) with the anti-cancer agent and the cardioprotective agent. Additionally, the present method can include the further step of determining a further level of therapeutic efficacy for the anti -cancer agent in combination with the cardioprotective agent. Similarly, cancer cells obtained or derived from the subject can be contacted or cultured simultaneously or sequentially with the anti-cancer agent and the cardioprotective agent.

[0087] Methods of treating cancer may be prophylactic, preventative or therapeutic and suitable for treatment of such diseases, disorders or conditions in mammals, particularly humans.

[0088] As used herein, “treating”, “treat” or “treatment” refers to a therapeutic intervention, course of action or protocol that at least ameliorates a symptom of such a disease, disorder or condition after said disease, disorder or condition and / or its symptoms have at least started to develop. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a cancer, or exhibits only early signs, for the purpose of decreasing the risk of developing a symptom, aspect, or characteristic of a cancer. A “therapeutic” treatment is one administered to a subject who exhibits at least one symptom, aspect, or characteristic of the cancer so as to cure, remediate or reverse, at least in part, and / or halt or delay the progression of said symptom, aspect, or characteristic. As used herein, “preventing”, “prevent” or “prevention” refers to therapeutic intervention, course of action or protocol initiated prior to the onset of a cancer and / or a symptom thereof so as to prevent, inhibit or delay the development or progression of such diseases, disorders or conditions or a symptom thereof. It is to be understood that such preventing need not be absolute to be beneficial to a subject.

[0089] The therapeutic agents (e.g., the anti-cancer agent and optionally the cardioprotective agent) or composition described herein may be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the frequency of administration is dependent on the preparation applied, the subject being treated, the severity of the disease, disorder or condition (e.g., cancer), and the manner of administration of the therapy or composition.

[0090] Any safe route of administration may be employed for administering an agent, inclusive of an anti-cancer agent and a cardioprotective agent, and the pharmaceutical compositions described herein. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra- articular, intramuscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be achieved by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids arid certain cellulose derivatives such as hydroxypropylmethyl cellulose, in addition, the controlled release may be achieved by using other polymer matrices, liposomes and / or microspheres. In particular examples, the agent or composition is capable of being or configured or adapted to be administered orally to a subject in need thereof. For such examples, the composition is suitably enterically coated.

[0091] Compositions of the present disclosure suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the disclosure, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water- in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the therapeutic agents of the disclosure with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

[0092] The above compositions or therapeutic agents may be administered in a manner compatible with the dosage formulation, and in such an amount as is effective to prophylactically and / or therapeutically treat the subject’s cancer and / or alleviate one or more symptoms associated therewith. The dose administered to a patient, in the context of the present disclosure, should be sufficient to achieve a beneficial response in a patient over time such as a complete or partial response or stable disease (e.g., little or no disease progression / metastasis). The quantity of the therapeutic agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the therapeutic agent(s) required to be administered will depend on the judgement of the clinician. The total dose required for each treatment may be administered by multiple doses or in a single dose. In any event, suitable dosages of the therapeutic agents described herein may be readily determined by those skilled in the art. Such dosages may be in the order of nanograms to milligrams of the therapeutic agents of the disclosure.

[0093] According to particular examples, a dose of the anti-cancer agent administered to the subject is at least partly determined by the level of therapeutic efficacy determined in step (b), as discussed in more detail herein. To this end, the dose of the anti-cancer agent administered to the subject is equivalent to or correlates with, for example, an effective amount or concentration of the anti-cancer agent determined in step (b) (e.g., a dose equivalent to an amount or concentration that has demonstrated a high level of therapeutic efficacy in the subject’s cancer cells of step (b)). Alternatively, or additionally, a dose of the anti-cancer agent administered to the subject can be at least partly determined by the level of cardiotoxicity determined in step (a), as described in more below. To this end, the dose of the anti-cancer agent administered to the subject is equivalent to or correlates with, for example, an amount or concentration of the anti-cancer agent determined in step (a) that demonstrates little, substantially no or no cardiotoxicity in the subject’s cardiac cells (e.g., a dose equivalent to an amount or concentration that has demonstrated a low level of cardiotoxicity in the subject’s cardiac cells of step (a)). In particular examples, the dose of the anticancer agent administered to the subject is equivalent to or correlates with an amount or concentration of the anti-cancer agent that has been determined: in step (a) to demonstrate little, substantially no or no cardiotoxicity in the subject’s cardiac cells; and in step (b) to demonstrate a high level of therapeutic efficacy in the subject’s cancer cells.

[0094] Suitably, the therapeutic agents disclosed herein (e.g., the anti-cancer agent and / or optionally the cardioprotective agent) can be administered to a subject suffering from a cancer, in a therapeutically effective amount sufficient to cure, or at least partially arrest said cancer and its complications (e.g., metastasis). The therapeutically effective dose level for any particular patient or subject, will depend upon a variety of factors familiar to one skilled in the art, including, for example: the disease, disorder or condition being treated and / or the severity of the disease, disorder or condition, the age, body weight, general health, sex and diet of the patient, the time of administration, the route of administration, the duration of the treatment, drugs used in combination or coincidental with the treatment, together with other related factors well known in medicine. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, gender, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, a patient's body surface area, whether the patient is undergoing therapy, and any specific contraindications.

[0095] In particular examples, the anti-cancer agent is administered, such as daily, weekly, fortnightly or monthly, for an appropriate period of time, such as 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 1 year etc, in an amount or dose of about 0.5 mg to about 500 mg (e.g., about 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480 or 500 mg or any range therein), or about 5 mg to about 200 mg, or about 10 mg to about 100 mg, or about 20 mg to about 80 mg, or about 25 mg to about 50 mg, or about 5 mg to about 500 mg, or about 5 mg to about 400 mg, or about 5 mg to about 300 mg, or about 5 mg to about 200 mg, or about 5 mg to about 100 mg, or about 5 mg to about 10 mg, or about 5 mg to about 20 mg, or about 5 mg to about 40 mg, or about 5 mg to about 60 mg.

[0096] Cardiotoxicity

[0097] The term “cardiotoxicity” refers to a broad range of adverse effects on heart structure and function induced by therapeutic molecules. It may emerge early in pre-clinical studies or become apparent later in the clinical setting. Cardiovascular toxicity described herein, inclusive of monitoring thereof, can include, but is not limited to, any one or more of increased QT duration, arrhythmias, myocardial ischemia, cardiovascular fibrosis, cardiovascular inflammation, myocarditis, hypertension, thromboembolic complications, myocardial dysfunction, cardiomyopathy, heart failure, left ventricular dysfunction, right ventricular dysfunction, systolic dysfunction, diastolic dysfunction, atrial flutter, atrial fibrillation, heart valve damage or dysfunction, vascular damage or dysfunction and any combination thereof. Further cardiac damage resulting from drug-induced cardiotoxicity can be one or more of degenerative changes in cardiomyocytes, loss of myofibrils, vacuolization of cytoplasm, mitochondrial damage, inflammatory cell infiltration, cardiomyocyte cell death (e.g., non-programmed or programmed or due to apoptosis, autophagy or fibrosis), myocyte cell replacement, interstitial cell changes, cardiac fibrosis, nitrosative stress, mitochondrial damage and stress, genotoxic stress (e.g., TOP2B- mediated stress), endoplasmic reticulum stress, dysregulation of ubiquitin-proteasome system, Ca2+ homeostatic impairment, myocardial progenitor cell damage, impaired expression of myofilament proteins and / or their cleavage, dysregulation of paracrine signalling (e.g., neuregulin- 1, nitric oxide, renin-angiotensin-aldosterone system), mitochondrial damage or iron accumulation.

[0098] Drug-induced toxicity, and more particularly cardiotoxicity, is suitably assessed in vitro or ex vivo by any means, such as cell-, tissue-, spheroid- or organoid-based assays, as described in more detail herein.

[0099] Suitably, the level of cardiotoxicity indicates or correlates with a particular likelihood or risk (e.g., a low, medium or high likelihood or risk) of cardiotoxicity for the anti-cancer agent in the subject. In some examples, subjects may be divided or stratified into particular risk groups, such as low risk, medium risk or high risk, based on their predicted levels of cardiotoxicity to an anti-cancer agent. In this regard, the predicted levels of cardiotoxicity may be compared to one or more pre-determined reference ranges or subranges of such levels. By way of example, subjects with cancer may be divided or stratified into low risk, medium or intermediate risk, or high risk groups according to a low risk subrange, a medium risk subrange, and a high risk subrange of a level of cardiotoxicity.

[0100] The term “low risk”, “low likelihood” or “low level of cardiotoxicity” in relation to cardiotoxicity, refers to a subject with a low or lower probability (e.g., less than about a 25%, 20%, 15%, 10% or 5% probability, likelihood or chance) of cardiotoxicity when administered the anti- cancer agent in question. The term “high risk”, “high likelihood” or “high level of cardiotoxicity” in relation to cardiotoxicity, refers to a subject with a higher probability (e.g., greater than about a 70%, 75%, 80%, 85%, 90% or 95% probability, likelihood or chance) of cardiotoxicity when administered the anti-cancer agent in question. The term “intermediate risk”, “intermediate likelihood” or “intermediate level of cardiotoxicity” in relation to cardiotoxicity, refers to a subject with an intermediate probability (e.g., a probability, likelihood or chance of between about 25% to about 70%, such as 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or any range therein) of cardiotoxicity when administered the anti-cancer agent in question.

[0101] It is envisaged that a subject’s levels of cardiotoxicity and therapeutic efficacy for the anticancer agent can be utilised to determine whether said subject with cancer can be, at least partly, treated with a low dose or reduced dose of the anti -cancer agent. The term “low dose” or “reduced dose” as used herein refers to a therapeutically effective dose of a treatment, such as an anti-cancer agent provided herein, whose dose is significantly or substantially less (e.g., at least about 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% etc less) than the usual, the standard or the conventional dose required to produce a therapeutic effect (e.g., an anti-cancer effect). Suitably, in such instances where a low dose of the anti-cancer agent is to be or is administered to the subject: (a) the level of cardiotoxicity of the anti -cancer agent indicates or correlates with a low likelihood of cardiotoxicity with that low dose of the anti-cancer agent; and / or (b) the level of therapeutic efficacy of the anti-cancer agent indicates or correlates with an intermediate or high likelihood of therapeutic efficacy with that low dose of the anti-cancer agent. In such instances, the level of cardiotoxicity of the anti-cancer agent may also indicate or correlate with an intermediate or high likelihood of cardiotoxicity with a standard dose of the anti-cancer agent.

[0102] Moreover, a subject’s levels of cardiotoxicity and therapeutic efficacy for the anti-cancer agent can be utilised to determine whether said subject with cancer can be, at least partly, treated with a high dose or increased dose of the anti -cancer agent. The term “high dose” or “increased dose” as used herein refers to a therapeutically effective dose of a treatment, such as an anti-cancer agent provided herein, whose dose is significantly or substantially more (e.g., at least about 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% etc more) than the usual, the standard or the conventional dose required to produce a therapeutic effect. Suitably, in such instances where a high dose of the anti-cancer agent is to be or is administered to the subject: (a) the level of cardiotoxicity of the anti-cancer agent indicates or correlates with a low likelihood of cardiotoxicity with that high dose of the anti-cancer agent; and / or (b) the level of therapeutic efficacy of the anti-cancer agent indicates or correlates with an intermediate or high likelihood of therapeutic efficacy with that high dose of the anti -cancer agent.

[0103] In certain examples, if the level of cardiotoxicity is equal to or higher than a reference or threshold level thereof (e.g., a mean or median level of cardiotoxicity in a population of patients administered the anti-cancer agent), the subject is to be administered a low or reduced dose of the anti-cancer agent. For some examples, if the level of cardiotoxicity is lower than the reference or threshold level thereof, the subject is to be administered a high, conventional or standard dose of the anti-cancer agent. In other examples, if (i) the level of cardiotoxicity is equal to or higher than a reference or threshold level thereof, the subject can be administered a high, conventional or standard dose of the anti-cancer agent and optionally a cardioprotective agent. In those instances, in which the level of cardiotoxicity is equal to or higher than a reference or threshold level thereof, the subject is suitably monitored for cardiotoxicity during administration of the anti-cancer agent and optionally the cardioprotective agent. In other examples, if the level of cardiotoxicity is equal to or higher than a reference or threshold level thereof, the subject is not administered the anticancer agent or administration of the anti -cancer agent is stopped or discontinued in the subject. In such examples, a further anti-cancer agent can be administered to the subject instead of the anticancer agent in question, such as a further anti-cancer agent that is predicted to demonstrate a low likelihood of cardiotoxicity and a high likelihood of therapeutic efficacy in the subject by the methods provided herein.

[0104] In particular examples, the level of cardiotoxicity is determined at least partly by detecting a level or presence of one or more indicators, biomarkers or readouts of cardiotoxicity, such as vascular damage, vascular dysfunction, cardiac cell viability, cardiac cell atrophy, cardiac cell necrosis, cardiac cell apoptosis, cardiac fibrosis, contractile dysfunction (systolic and / or diastolic dysfunction), electrophysiological dysfunction (e.g., calcium signalling and / or action potential dysfunction), mitochondrial damage, oxidative stress and inflammation in the cardiac cells of the subject. Such indicators of cardiotoxicity may be assessed in the cardiac cells by any means known in the art, such as those provided below. Cell viability is a measure of the proportion of live, healthy cells within a population and exemplary cell viability assays include an alamar blue assay, an MTT / MTS assay, calcein-AM (live cells) staining, a hsTNT / cTNT assay and a secreted reporter assay. Cardiac cell necrosis and apoptosis can be measured by the detection of apoptotic and necrotic markers, ethidium homodimer (dead cells) staining, lineage-specific cell death reporters, or indirectly by measuring a decrease in cell viability. Cardiac cell toxicity can be measured by toxicity ratio (dead / live) measurements based on calcein-AM (live cells) and ethidium homodimer (dead cells) staining. Contractile function can be assessed by any assay that measures, for example, contraction frequency, fractional shortening %, conduction velocity based on action potential and calcium staining, contractile strength measurement and the amplitude, contractile velocity, total signal under a transient curve and relaxation time for diastolic function. Exemplary mitochondrial activity or function assays can measure or assess disruption of mitochondrial function, which may be detected using a variety of fluorescence-based assays including measurements of mitochondrial calcium, superoxide, mitochondrial permeability transition, and membrane potential. Cardiac fibrosis assays can include immunostaining (e.g., antibodies against collagen) or histological (PicroSirius Red and / or Masson’s Trichrome staining) staining for fibrosis or fibrotic markers, proteomics analyses, or real time qPCR of fibrotic marker transcripts, including transcriptomics analyses (bulk RNAseq, scRNAseq and spatial transcriptomics). Inflammation may be assessed by monitoring the transcripts of inflammatory markers or infiltration / activation of inflammatory cells by methods well-known in the art. Assays to determine oxidative stress may measure levels of toxic reactive oxygen species or levels of cellular antioxidants.

[0105] Suitably, determining the level of cardiotoxicity comprises contacting the cardiac cells with one or more doses or concentrations (e.g., with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc., different doses or concentrations) of the anti-cancer agent, such as for a time and under conditions to determine a level of cardiotoxicity thereof. For some examples, the cardiac cells are contacted with a dose range of the anti-cancer agent (e.g., to determine a dose sensitivity, a dose response curve, an IC50 or an EC50). The term “dose range” as used herein refers to an upper and a lower limit of an acceptable variation of the amount of agent specified. Typically, a dose or a dose range of the anticancer agent utilised in determining the level of cardiotoxicity and / or the level of therapeutic efficacy (e.g., an in vitro dose or dose range) is equivalent to or correlates with a dose, an amount, or specified dose range that can be administered to patients undergoing treatment (e.g., an in vivo dose or dose range).

[0106] Accordingly, the present method may be utilised to determine or select a dose of the anticancer agent from a range or plurality of doses thereof tested, which is appropriate or optimal for the subject in question. By way of example, the dose of the anti-cancer agent utilised or administered to the subject is suitably predicted to have an intermediate or high likelihood of therapeutic efficacy for the subject’s cancer and optionally a low or intermediate likelihood of cardiotoxicity in the subject.

[0107] It is contemplated, however, that the dose of the anti-cancer agent utilised or administered to the subject may have been predicted to have a high likelihood of cardiotoxicity in the subject. In such instances, the subject may be monitored for one or more clinical signs or symptoms of cardiotoxicity (such as one or more of those in vivo indicators or types of cardiotoxicity described herein which equates to that indicator or type of cardiotoxicity observed in vitro in the cardiac cells of the subject) during administration of the anti-cancer agent, as described in more detail herein. Additionally or alternatively, the subject may be administered a cardioprotective agent, such as those described herein, in an attempt to reduce or minimise the risk or likelihood of cardiotoxicity in the subject, particularly in those instances in which the cardioprotective agent, in combination with the anti-cancer agent, has been demonstrated to reduce the level of cardiotoxicity for the anti-cancer agent in the cardiac cells derived from the subject.

[0108] The results of in vitro or ex vivo cardiotoxicity testing described herein may be confirmed or monitored in a subject at one or more time points (e.g., about 1 day, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 12 months, 24 months etc) during administration of the anti-cancer agent in question by in vivo imaging techniques (e.g., echocardiography, cardiac magnetic resonance imaging), electrophysiological techniques and biomarkers. This may particularly be performed in those patients in which the determined level of cardiotoxicity indicates or correlates with an intermediate or high likelihood of cardiotoxicity for the anti-cancer agent in the subject, but may also be performed in those subjects determined to be of low risk of cardiotoxicity developing with administration of the anti-cancer agent. Exemplary in vivo biomarkers of cardiotoxicity include QTc prolongation, electrophysiological arrhythmias, circulating troponin c, heart rate, blood pressure, circulating lipids, C-reactive protein (CRP), brain or B-type natriuretic peptide (BNP), ex vivo platelet aggregation and imaging biomarkers.

[0109] Similarly, in vitro or ex vivo cardiotoxicity testing as described herein may continue or be repeated at one or more time points (e.g., about 1 day, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 12 months, 24 months etc) during administration of the anti -cancer agent. In particular examples, the present methods further include the step of determining a further level of cardiotoxicity for the anti-cancer agent in the cardiac cells at a further time point following or during administration of the anti-cancer agent. For such examples, the cardiac cells may be cultured in a media comprising blood, serum or plasma derived from the subject at or around the further time point so as to attempt to mimic physiological conditions in the subject at the further time point.

[0110] As used herein, the terms “little or no toxicity”, “little or no cardiotoxicity”, “low level of cardiotoxicity” refer to a level or incidence, including a predicted level or incidence, of toxicity and cardiotoxicity respectively for an anti -cancer agent of the present disclosure administered at a therapeutically effective dose (e.g., about 1 mg / day, about 5 mg / day, about 10 mg / day, about 20 mg / day, about 30 mg / day, about 40 mg / day, about 50 mg / day, about 60 mg / day, about 70 mg / day, about 80 mg / day, about 90 mg / day, about 100 mg / day, about 200 mg / day, about 500 mg / day or about 1000 mg / day of the agent) that typically is essentially equivalent to (e.g., no more than about 1%, 5% or 10% above) that level or incidence of toxicity or cardiotoxicity observed in patients when using a control or placebo only (e.g., a vehicle, such as PBS) that does not contain the anticancer agent.

[0111] Similarly, the terms “intermediate level of cardiotoxicity” or “medium level of cardiotoxicity” refer to a level or incidence, including a predicted level or incidence, of toxicity and cardiotoxicity respectively for an anti -cancer agent of the present disclosure administered at a therapeutically effective dose that is moderately higher than (e.g., more than about 15%, 20%, 25%, 30%, 35%, 40% or 45% above) that level or incidence of toxicity or cardiotoxicity observed in patients when using a control or placebo only that does not contain the anti-cancer agent. Moreover, the term “high level of cardiotoxicity” refer to a level or incidence, including a predicted level or incidence, of toxicity and cardiotoxicity respectively for an anti -cancer agent of the present disclosure administered at a therapeutically effective dose that is significantly higher than (e.g., more than about 50%, 60%, 70%, 80% or 90% above) that level or incidence of toxicity or cardiotoxicity observed in patients when using a control or placebo only that does not contain the anti-cancer agent.

[0112] It is further contemplated that the cardiotoxicity described herein can be of more than one type or form for each anti-cancer agent, which can vary in sensitivity and expression between individual patients. This can include, for example hypertension, dyslipidaemia, hypertriglyceridemia, hyperglycaemia, QT interval prolongation, heart block, ventricular arrhythmias, atrial arrhythmias, AF, Heart failure, vascular toxicity, atherosclerosis, arteritis, vasculitis, thrombosis, arterial occlusion, arterial dissection, pulmonary hypertension, myocardial infarction, stroke, Takotsubo cardiomyopathy and related conditions as will be known to one skilled in the art.

[0113] Moreover, the level of cardiotoxicity determined herein may be based on the determination or detection of cardiotoxicity at any time during or after contacting or culturing the subject’s cardiac cells with the anti -cancer agent and optionally the cardioprotective agent. Accordingly, the level of cardiotoxicity may be based on the detection or determination of any acute cardiotoxicity, chronic cardiotoxicity or delayed cardiotoxicity present in the cardiac cells of the subject with administration of the anti-cancer agent thereto.

[0114] In some examples, the methods described herein provide a reduction of at least about 25%, at least about 50%, at least about 75% or at least about 80%, in probability of cardiotoxicity occurring or being detected during the treatment period with the anti-cancer agent. In other examples, the current methods prevent a change of at least about 25%, at least about 50%, at least about 75% or at least about 80% of the concentration, amount or level of one or more biomarkers, indicators or readouts of cardiotoxicity.

[0115] Therapeutic efficacy

[0116] Herein, the term “therapeutic efficacy” refers to the capacity or ability of a substance, such as an anti-cancer agent, to achieve a certain therapeutic effect, such as a reduction in cancer volume and / or progression (e.g., local or distant spread or metastasis).

[0117] A level of therapeutic efficacy can be measured by determining or predicting the extent in which an anti-cancer agent can achieve the desired effect in the subject. By way of example, the level of therapeutic efficacy of the anti-cancer agent may be based at least in part on genetic information or genetic analysis (e.g., mutational status of one or more genetic markers of drug response) derived from the subject and / or the subject’s cancer. In other examples, the level of therapeutic efficacy of the anti-cancer agent may be based at least in part on the level of one or more biomarkers (e.g., levels of one or more biomarkers of drug response) in a biological sample (e.g., a blood sample, a biopsy sample) obtained or derived from the subject. Further exemplary methods or means of determining or predicting the level of therapeutic efficacy for the anti-cancer agent may include determining or detecting the presence or absence of particular clinical features, such as cancer stage and / or grade, metastasis, local spread, regional spread, lymph node involvement, para-neoplastic features, cachexia, nausea, pain, cancer cell viability, an angiogenic response to an anticancer drug, tumour growth, tumour size and cancer stem cells. In further examples, a level of therapeutic efficacy is determined, at least partly, by a predictive model of drug response that is based on one or more genetic markers, biomarkers and clinical parameters from the subject, such as those described above.

[0118] It is envisaged that the method of the present aspect may be performed in relation to cells in vitro, in vivo or ex vivo. In some examples, the method is performed in vitro, such as with cancer cells isolated from a subject or cardiac organoid culture, as described herein. In other examples, the present method is performed in vivo in a subject.

[0119] In view of the above, step (b) of the methods described herein suitably includes determining a level of therapeutic efficacy for the anti-cancer agent in cancer cells obtained or derived from the subject’s cancer. Accordingly, the methods described herein may include the initial or earlier step of isolating the cancer cells from the subject, such as by way of a biopsy or other biological sample.

[0120] For the purposes of the present disclosure, by “isolated” is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material (e.g., cardiac cells and cancer cells) may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.

[0121] Suitably, the cancer cells comprise a 2D culture (e.g., a cancer cell line), a 2D co-culture (e.g., cancer cells with stromal cells), a 2.5D culture (e.g., cells growing on top of a layer of an extracellular matrix (ECM) protein or a hydrogel), a 3D culture (e.g., cancer organoids or tumoroids, such as a patient-derived organoid) or a patient-derived xenograft (PDX). As such, the methods described herein may include the initial or earlier step of preparing or generating a 2D culture, a 2D co-culture, a 2.5D culture, a 3D culture or an animal -based cancer model (e.g., a patient-derived xenograft, administration or transplantation of a cancer cell line to an animal model) from the cancer cells derived or obtained from the subject, such as by those methods well known in the art.

[0122] According to some examples, the cancer cells are or comprise a cancer cell line. The term “cancer cell line” as used herein, refers to a line of cells established from a primary cancer cell, in which all cells possess the same properties. These cells typically, but not always, grow rapidly in culture when supplemented with appropriate growth factors, often foetal animal serum. The cancer cell line may or may not be immortalised.

[0123] In other examples, the cancer cells are or comprise a patient-derived xenograft (PDX). The terms “patient-derived xenograft” and “PDX” refer to a cancer model in which tissue or cells from a patient's tumour are implanted into an immunodeficient or humanized mouse. The PDX model is used to create an environment that allows for the natural growth of the cancer, its monitoring, and the corresponding treatment assessment of the subject from which the PDX model has been derived.

[0124] In some examples, the cancer cells are or comprise a patient-derived organoid (PDO). Patient-derived organoids (PDOs) are three-dimensional cell cultures that mimic the structure and function of human tissues. Such PDOs may also be implanted into an immunodeficient or humanized mouse. In contrast to patient-derived xenografts, organoids can typically be established and expanded with high efficiency from primary patient material. On the other hand, xenografts generally retain tumour-stroma interactions, which may contribute to tumorigenesis.

[0125] Therapeutic efficacy may be determined by routine practice, for example, efficacy may be assessed in the treatment of any of the aforementioned models. By way of example, for an animator rodent-based cancer model, any anti-cancer agent that leads to a reduction in tumour size, a slowing or slowing of the growth rate of a tumour, or preventing or minimising metastasis of the tumour, represents an effective treatment (e.g., a medium or high level of therapeutic efficacy). In other examples, the level of therapeutic efficacy may be assessed in cell-based or in vitro models, such as those described above, by assessing levels of one or more markers, indicators or readouts of therapeutic efficacy, such as cell proliferation, cell apoptosis, cell death, cell detachment / adhesion, cell invasion / migration and cell viability in the presence of the anti-cancer agent. Such cellular indicators of therapeutic efficacy may be assessed by any means or method known in the art, including, but not limited to, flow cytometry (e.g., trypan blue, annexin V staining, propidium iodide), fluorescent based cell detection assays (e.g., Calcein-AM, Ethidium Homodimer, Mitotracker Red), luminescent based detection assays (e.g., Cell-Titer Gio), spectrophotometry based detection assays (e.g., crystal violet, MTS / MTT assays such as Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay), a scratch wound assay, a boyden chamber assay and a cell invasion assay using fluorescent detection of cell invasion (e.g., activin, serum).

[0126] An exemplary measure for determining the level of therapeutic efficacy of an anti-cancer agent in vitro or preclinically is an IC50 value or an EC50 value. Accordingly, the level of therapeutic efficacy of the anti-cancer agent may be determined at least partly by culturing the cancer cells of the subject in the presence of one or more concentrations of the anti-cancer agent, such as for a time and under conditions to determine the level of therapeutic efficacy thereof. As used herein, the term “IC50” refers to the half maximal inhibitory concentration of an anti-cancer agent in inhibiting a biological or biochemical function (e.g., cell proliferation, cell invasion / migration, cell viability) of a cancer or cancer cell, such as that derived from the subject. This quantitative measure indicates how much of a particular anti-cancer agent is needed to inhibit a given biological process of the subject’s cancer (e.g., cell proliferation) by half. In other words, it is the half maximal (50%) inhibitory concentration (IC) of a substance (50% IC, or IC50). As used herein, the term "EC50" is intended to mean an amount, concentration or dosage of an anticancer agent that achieves a 50% of its maximal effect. Once the level of therapeutic efficacy has been determined for the anti-cancer agent, this may then be utilised to determine or estimate a therapeutically effective amount or effective amount thereof for use in treating the subject’s cancer. In the present context, the term “therapeutically effective amount” or “effective amount” indicates that a compound or amount of the compound when administered is, or is predicted to be, sufficient or effective to prevent, alleviate, or ameliorate one or more symptoms of a disease, disorder or medical condition being treated (e.g., cancer), and / or to prolong the survival of the subject being treated. The therapeutically effective amount will vary depending on the compound, the disease, disorder or condition and its severity and the age, weight, etc., of the mammal to be treated. In particular examples though, the therapeutically effective amount suitably reduces the number of cancer cells, reduces the tumour size, inhibits at least partially cancer cell infiltration into peripheral organs and tumour metastasis, inhibit at least partially tumour growth and / or relieve or ameliorate at least partially one or more of the symptoms associated with the subject’s cancer. In some examples, an effective amount of the anti -cancer agent is sufficient to produce a partial response in the subject. In other examples, an effective amount of the anti -cancer agent is sufficient to produce a complete response in the subject. Referring to the cancer cells, cardiac cells and methods described herein, the term “effective amount” can refer to an amount of an agent, such as an anti-cancer agent or a cardioprotective agent, sufficient to have a physiological, biological and / or therapeutic effect (e.g., inhibiting cancer cell proliferation and / or migration, inhibiting cardiotoxicity in cardiac cells) in respect of such cells in vitro. Such an effective amount can be determined by those methods and assays well known in the art, inclusive of those provided herein.

[0127] Suitably, the level of therapeutic efficacy indicates or correlates with a particular likelihood (e.g., a low, medium or high likelihood) of a positive therapeutic response or drug responsiveness in relation to the subject’s cancer with administration of the anti-cancer agent, and more particularly a therapeutically effective amount thereof, to the subject.

[0128] In some examples, specific anti-cancer agents may be divided or stratified into particular groups based upon their predicted therapeutic efficacy in the subject, such as low likelihood, medium likelihood or high likelihood. In this regard, the calculated levels of therapeutic efficacy may be compared to one or more pre-determined reference ranges or subranges of such levels. By way of example, anti-cancer agents may be divided or stratified into low likelihood, medium or intermediate likelihood, or high likelihood groups according to a low likelihood subrange, a medium likelihood subrange, and a high likelihood subrange of the level of therapeutic efficacy.

[0129] The term “low likelihood” in relation to therapeutic efficacy may also refer to a cancer or patient with a lower or low probability (e.g., less than about a 25%, 20%, 15%, 10% or 5% probability, likelihood or chance) of at least partly responding, such as a complete or partial response, to the anti-cancer agent. The term “high likelihood” in relation to therapeutic efficacy may also refer to a cancer or patient with a higher or high probability (e.g., greater than about a 70%, 75%, 80%, 85%, 90% or 95% probability, likelihood or chance) of at least partly responding, such as a complete or partial response, to the anti -cancer agent. The term “intermediate likelihood” in relation to therapeutic efficacy may also refer to a cancer or patient with an intermediate probability (e.g., a probability, likelihood or chance of between about 25% to about 70%, such as 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or any range therein) of at least partly responding to an anti-cancer treatment.

[0130] Anti-cancer agents

[0131] Generally, drugs (e.g., small molecules) or biomolecules (e.g., antibodies, inhibitory nucleic acids such as siRNA, inhibitory peptides, such as cyclic peptides) are referred to herein as “anti-cancer therapeutic agents” or “anti-cancer agents”. It is envisaged that the term “anti-cancer agent” can include a single anti-cancer agent (i.e., a monotherapy) or alternatively relate to two or more (e.g., 2, 3, 4, 5, etc) anti-cancer agents (i.e., a combination therapy), such as those described herein. In this regard, it will be appreciated that the response (i.e., cardiotoxicity and / or therapeutic response) of a subject’s cancer cells and / or cardiac cells to a combination of two or more different anti-cancer agents, such as those described herein, may differ (e.g., demonstrate a greater or lesser risk of cardiotoxicity) from what may be expected or determined by testing these anti-cancer agents in isolation, such as by the methods described herein.

[0132] Suitably, the anti-cancer agent described herein can be administered as a composition comprising such an agent or agents, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0133] By “pharmaceutically-acceptable carrier, diluent or excipient” is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water,

[0134] A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ. USA, 1991), which is incorporated herein by reference.

[0135] Suitably, the anti-cancer agent is selected from the group consisting of: a chemotherapeutic agent, a molecularly targeted agent, a T cell expressing a chimeric antigen receptor (CAR-T cell), an antibody or antigen-binding fragment thereof, an antibody-drug conjugate (ADC), an angiogenesis inhibitor, a hormone therapy, an immunotherapeutic agent, radiotherapy, a radiopharmaceutical and any combination thereof. In particular examples, the anti-cancer agent is or comprises one or more of a chemotherapeutic agent, a molecularly targeted agent, a hormone therapy and an immunotherapeutic agent, including any combination thereof.

[0136] Referring to some examples, the anti-cancer agent is or comprises a chemotherapeutic agent. As generally used herein, the term “chemotherapy” or “chemotherapeutic agent” broadly refers to a treatment or agent with a cytostatic or cytotoxic agent (i.e., a compound) to reduce or eliminate the growth or proliferation of undesirable cells, such as cancer cells. Accordingly, the terms can refer to a cytotoxic or cytostatic agent used to treat a proliferative disorder, for example cancer. The cytotoxic effect of the agent can be, but is not required to be, the result of one or more of nucleic acid intercalation or binding, DNA or RNA alkylation, inhibition of RNA or DNA synthesis, the inhibition of another nucleic acid-related activity (e.g., protein synthesis), or any other cytotoxic effect.

[0137] Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracil mustard; aziridines such as thiotepa; methanesulphonate esters such as busulfan; nitroso ureas such as carmustine, lomustine, and streptozocin; platinum complexes such as cisplatin and carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin and lipoplatin; bioreductive alkylators such as mitomycin, procarbazine, dacarbazine and altretamine); DNA strand-breakage agents (e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, and teniposide); DNA minor groove binding agents (e.g., plicamydin); antimetabolites (e.g., folate antagonists such as methotrexate and trimetrexate; pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine; purine antagonists such as mercaptopurine, 6-thioguanine, fludarabine, pentostatin; asparginase; and ribonucleotide reductase inhibitors such as hydroxyurea); and tubulin interactive agents (e.g., vincristine, vinblastine, and paclitaxel (Taxol)).

[0138] In other examples, the anti -cancer agent is or comprises a molecularly targeted therapy. As used herein, “molecularly targeted therapy” or “molecularly targeted therapeutic agent” refers to a therapy that targets a particular class of proteins involved in cancer growth or signalling. It is envisaged that such agents may include antibodies or fragments thereof and small molecules.

[0139] In some examples, the anti -cancer agent described herein is or comprises an inhibitor of a kinase, such as a tyrosine kinase or a serine-threonine kinase. The term “tyrosine kinase” refers to enzymes which are capable of transferring a phosphate group from ATP to a tyrosine residue in a protein. Phosphorylation of proteins by tyrosine kinases is an important mechanism in signal transduction for regulation of enzyme activity and cellular events such as cell survival or proliferation. Tyrosine kinases include receptor tyrosine kinases and non-receptor tyrosine kinases. AATK, ABL, ABL2, ALK, AXL, BLK, BMX, BTK, CSF1 R, CSK, DDR1, DDR2, EGFR, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6, ERBB2, ERBB3, ERBB4, FER, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT1, FLT3, FLT4, FRK, FYN, GSG2, HCK, IGF1R, ILK, INSR, INSRR, IRAK4, ITK, JAK1, JAK2, JAK3, KDR, KIT, KSR1, LCK, LMTK2, LMTK3, LTK, LYN, MATK, MERTK, MET, MLTK, MST1R, MUSK, NPR1, NTRK1, NTRK2, NTRK3, PDGFRA, PDGFRB, PLK4, PTK2, PTK2B, PTK6, PTK7, RET, R0R1, R0R2, ROS1, RYK, SGK493, SRC, SRMS, STYK1, SYK, TEC, TEK, TEX14, TIE1, TNK1, TNK2, TNNI3K, TXK, TYK2, TYR03, YES1, and ZAP70.

[0140] According to various examples, the anti -cancer agent is or comprises an immunotherapeutic agent. Insofar as they relate to cancer, immunotherapy or immunotherapeutic agents use or modify the immune mechanisms of a subject so as to promote or facilitate treatment of a cancer. In this regard, immunotherapy or immunotherapeutic agents used to treat cancer include cell -based therapies, antibody therapies (e.g., anti-PDl or anti-PDLl antibodies) and cytokine therapies. These therapies all exploit the phenomenon that cancer cells often have subtly different molecules termed cancer antigens on their surface that can be detected by the immune system of the cancer subject. Accordingly, immunotherapy is used to provoke the immune system of a cancer patient into attacking the cancer's cells by using these cancer antigens as targets.

[0141] Non-limiting examples of immunotherapy or immunotherapeutic agents include adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, BMS-936559, brentuximab, certolizumab, cituximab, daclizumab, eculizumab, ibritumomab, infliximab, ipilimumab, lambrolkizumab, mepolizumab, MPDL3280A muromonab, natalizumab, nivolumab, ofatumumab, omalizumab, pembrolizumab, pexelizumab, pidilizumab, rituximab, tocilizumab, tositumomab, trastuzumab, ustekinumab, abatacept, alefacept and denileukin diftitox. In particular preferred embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor, such as an anti-PDl antibody (e.g., pidilizumab, nivolumab, lambrolkizumab, pembrolizumab), an anti- PDL1 antibody (e.g., BMS-936559, MPDL3280A) and / or an anti-CTLA4 antibody (e.g., ipilimumab).

[0142] In further examples, the anti -cancer treatment is or comprises a hormone therapy. As used herein, the term “hormone therapy” refers to a therapy for the treatment of a cancer that deprives a tumor of a hormone, (e.g., an androgen, such as testosterone, or an oestrogen) or hormone activity in the subject. Suitable hormone therapies are known in the art and include but are not limited to, hormonal agents (e.g., estrogens; conjugated estrogens; ethinyl oestradiol; diethylstilbesterol; chlortrianisen; idenestrol; progestins such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and androgens such as testosterone, testosterone propionate, fluoxymesterone, and methyltestosterone); adrenal corticosteroids (e.g., prednisone, dexamethasone, methylprednisolone, and prednisolone); luteinizing hormone releasing agents or gonadotropin-releasing hormone antagonists (e.g., leuprolide acetate and goserelin acetate); and antihormonal antigens (e.g., tamoxifen, anti-androgen agents, such as flutamide; and anti-adrenal agents such as mitotane and aminoglutethimide).

[0143] For certain examples, the anti-cancer treatment is or comprises an antibody or a fragment thereof. The term “antibody” is used in the context of the present disclosure to refer to immunoglobulin molecules immunologically reactive with a particular antigen and includes both polyclonal and monoclonal antibodies. The term also includes native and genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). The term “antibody” also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab', F(ab')2, Fab, Fv and rlgG as discussed in Pierce Catalogue and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rdEd., W.H. Freeman & Co., New York (1998). The term antibody also includes multispecific molecules, such as bivalent or bispecific molecules. Examples of bivalent and bispecific molecules are described in Kostelny et al. (1992) J Immunol 148: 1547; Pack and Pluckthun (1992) Biochemistry 31 : 1579; Hollinger et al., 1993, supra, Gruber et al. (1994) J. Immunol. :5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.

[0144] An “antibody fragment” or “antigen binding fragment” of an antibody comprises one or more variable regions of an intact antibody. Examples of antibody fragments include Fab, Fab1, F(ab')2 and Fv fragments; diabodies; triabodies; tetrabodies; dimerisation-activated demibodies (e.g., WO / 2007 / 062466); linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments. For example, the term antigen binding fragment may be used to refer to recombinant single chain Fv fragments (scFv), as well as divalent (di-scFv) and trivalent (tri-scFV) forms thereof.

[0145] Referring to other examples, the anti-cancer treatment is or comprises a CAR-T cell. A “CAR-T cell” is a T cell which has been transduced with at least one CAR. CAR-T cells can be mono, dual, or tandem CAR-T cells. CAR-T cells can be autologous, meaning that they are engineered from a subject's own cells, or allogeneic, meaning that the cells are sourced from a healthy donor, and in many cases, engineered so as not to provoke a host-vs-graft or graft-vs-host reaction.

[0146] In various examples, the anti-cancer treatment is or comprises an ADC. The term “ADC” or “antibody-drug conjugate” has its usual scientific meaning known to those skilled in the art, and here refers to a class of biopharmaceutical drugs designed as a targeted therapy to treat, for example, cancer. Unlike chemotherapy, ADCs are intended to attack and destroy cancer cells without affecting healthy cells of the patient. ADCs are composed of an antibody linked to a biologically active cytotoxic (anti-cancer) payload or drug. ADCs combine the targeting capabilities of monoclonal antibodies with the cancer-killing capabilities of cytotoxic drugs. They are designed with the intention of discriminating between healthy cells and diseased tissues, for example, cancer cells in a cancer.

[0147] Referring to certain examples, the anti-cancer agent is or comprises an angiogenesis inhibitor. The term “angiogenesis inhibitor,” as used herein, refers to any agent or molecule that reduces or inhibits the formation of new blood vessels in a mammal. With regard to cancer therapy, the angiogenesis inhibitor suitably reduces or inhibits the formation of new blood vessels in or on a tumour, preferably in or on a solid tumour.

[0148] Referring to certain examples, the anti-cancer agent or treatment is radiotherapy or radiation. In this regard, the present methods may provide a risk or likelihood of radiotherapy administration to an area of the chest (e.g., mantle radiotherapy) of the subject in question resulting in cardiotoxicity (e.g., radiation-induced damage to the cardiovascular system, such as coronary damage or disease, valvular damage or disease, pericardial damage or disease, myocardial damage or disease, conduction tissue damage or disease and autonomic nervous system damage or disease).

[0149] In view of the above and in some examples, the anti-cancer agent or treatment is a radiopharmaceutical. The term “radiopharmaceutical” broadly speaking refers to a medicinal compound that has a radioactive component. Radiopharmaceuticals are used in the field of nuclear medicine as either radioactive tracers in medical imaging or in therapy e.g. radiotherapy, brachytherapy. The radiopharmaceutical may comprise a biological targeting molecule which is labelled with a radioisotope.

[0150] In particular examples, the anti-cancer agent is selected from the group consisting of: an anthracycline chemotherapeutic agent (e.g., Doxorubicin, Epirubicin, Daunorubicin, Idarubicin), a taxane chemotherapeutic agent (e.g., paclitaxel), an alkylating chemotherapeutic agent (e.g., cisplatin, cyclophosphamide, ifosfamide, mitomycin), a HER2-targeted or anti-HER2 agent or inhibitor (e.g., Trastuzumab (Herceptin), Trastuzumab deruxtecan, Trastuzumab emtansine, Pertuzumab, T-DM1, Lapatinib, Neratinib, Tucatinib), a fluoropyrimidine chemotherapeutic agent (e.g., capecitabine, floxuridine, and 5 -fluorouracil (5-FU)), a VEGF-targeted or anti-VEGF agent or inhibitor (e.g., Bevacizumab, Sunitinib, Sorafenib, Pazopanib, Axitinib, Regorafanib, Cabozantinib, Lenvatinib, Vandetinib, Tivozanib), a BTK inhibitor (e.g., Ibrutinib, Acalabrutinib, Zanubrutinib), a proteosomal inhibitor (e.g., Bortezomib, Carfilzomib, Ixazomib), a BCR-Abl inhibitor (e.g., Imatinib, Nilotinib, Dasatinib, Bosutinib, Ponatinib, Asciminib), a Bcl2 inhibitor (e.g., Venetoclax) a Flt3 inhibitor (e.g., Gilteritinib) an ALK inhibitor (e.g., Crizotinib, Brigatinib, Lorlatinib, Alectinib) an EGFR inhibitor (e.g., Osimertinib), a CDK 4 / 6 inhibitor (e.g., Palbociclib, Riboci clib), an immune checkpoint inhibitor (e.g., a PD-1 inhibitor, such as Nivolumab and Pembrolizumab, a CTLA-4 inhibitor, such as Ipilimumab, a PD-L1 inhibitor, such as Atezolizumab, Durvalumab and Avelumab), a cell therapy (e.g., a CAR-T cell, a tumourinfiltrating lymphocyte (TIL)), an androgen deprivation agent (e.g., Goserelin, Leuprolide, Bicalutamide, Enzalutamide, Abiraterone), a Raf-MEK pathway inhibitor (e.g., a Raf inhibitor, such as Debrafanib and Vemurafenib, a MEK inhibitor, such as Trametinib), a PI3K inhibitor (e.g., Alpelisib), a RET inhibitor (e.g., Pralsetinib, Selpercatinib) a ROS kinase inhibitor (e.g., Entrectinib), a NTRK Fusion inhibitor (e.g., Larotrectinib) and any combination thereof. To this end, it is envisaged that combinations (e.g., 2, 3, 4, 5 etc) of such agents, used either simultaneously or sequentially, may be utilised in the methods described herein.

[0151] It will be appreciated that the therapeutic effect of the various anti-cancer agents described herein need not be absolute to elicit a biological effect, such as treatment of the subject’s cancer. Accordingly, therapeutic efficacy provided by an anti -cancer agent described herein can be partial or incomplete (e.g., a readout, indicator or biomarker of cancer progression in the subject is reduced by about 20%, 30%, 40%, 50%, 60% or 70%, 80%, 90%, 95%, 96%, 97%, 98% and 99%, including any intermediate value therebetween with administration of an anti-cancer agent as described herein).

[0152] Cardioprotective agents Suitably, the methods described herein further include the step of culturing the cardiac cells (and optionally the cancer cells) in the presence of a cardioprotective agent and the anti-cancer agent, such as for a time and under conditions to determine a level of cardiotoxicity thereof. To this end, the method may include determining whether contacting or culturing the cardiac cells with a cardioprotective agent and the anti-cancer agent modulates, or more particularly reduces, the level of cardiotoxicity for the anti -cancer agent in the subj ect. It is contemplated that the cardiac cells may be contacted or cultured with the anti-cancer agent and the cardioprotective agent simultaneously or sequentially (i.e., culturing the cardiac cells in the presence of the anti-cancer agent and then the cardioprotective agent or culturing the cardiac cells in the presence of the cardioprotective agent and then the anti-cancer agent).

[0153] For some examples, the present methods include culturing the cardiac cells with the anticancer agent alone (i.e., no cardioprotective agent) and also the anti -cancer agent in combination with one or more cardioprotective agents. Suitably, this allows for the selection of a cardioprotective agent that demonstrates a reduction in the level of cardiotoxicity when the subject’s cardiac cells are cultured in combination with the anticancer agent versus that when cultured with the anti-cancer agent alone.

[0154] Moreover, in some examples, the methods disclosed herein further include the step of administering to the subject a therapeutically effective amount of a cardioprotective agent in combination with the anti-cancer agent. As such, the anti -cancer agent may be administered alone (i.e., monotherapy) or alternatively be administered in combination with the cardioprotective agent which aims to at least partly treat or prevent any cardiotoxicity described herein associated with administration of the anti-cancer agent. Accordingly, the cardioprotective agent is suitably administered in combination with the anti-cancer agent when the level of cardiotoxicity determined for the anti-cancer agent indicates or correlates with a medium or high likelihood of cardiotoxicity in the subject (and optionally demonstrates a decreased level of cardiotoxicity when the subject’s cardiac cells are cultured with the anti -cancer agent and the cardioprotective agent). It is contemplated, however, that the cardioprotective agent may still be administered in combination with the anti-cancer agent when the level of cardiotoxicity determined for the anticancer agent indicates or correlates with a low likelihood of cardiotoxicity in the subject. Additionally, for such examples, the level of therapeutic efficacy suitably indicates or correlates with an intermediate or high likelihood of therapeutic efficacy of the anti-cancer agent in the subject.

[0155] It is envisaged that the present methods may further include the step of culturing the cardiac cells and / or the cancer cells of the subject in the presence of a cardioprotective agent and the anti- cancer agent. To this end a further level of cardiotoxicity and optionally a further level of therapeutic efficacy for the anti -cancer agent in the presence of the cardioprotective agent may be determined, such as by those methods disclosed herein. For such examples, the current methods may further include the step of determining whether contacting the cardiac cells with the cardioprotective agent in combination with the anti-cancer agent modulates, or more particularly reduces, the level of cardiotoxicity for the anti-cancer agent in the subject. Optionally, the current methods can further include the step of determining whether contacting the cardiac cells with the cardioprotective agent in combination with the anti-cancer agent modulates the level of therapeutic efficacy of the anti-cancer agent in the subject. In particular examples, the cardioprotective agent is administered in combination with the anti-cancer agent when contacting the cardiac cells with the cardioprotective agent in combination with the anti-cancer agent reduces the level of cardiotoxicity for the anti -cancer agent in the cardiac cells.

[0156] Therefore, in particular examples, the methods disclosed herein further include the step of administering to the subject a therapeutically effective amount of a cardioprotective agent in combination with the anti -cancer agent. Suitably, for such examples:

[0157] (a) the level of cardiotoxicity determined for the anti-cancer agent indicates or correlates with an intermediate or high likelihood of cardiotoxicity in the subject;

[0158] (b) the level of therapeutic efficacy suitably indicates or correlates with an intermediate or high likelihood of therapeutic efficacy of the anti-cancer agent in the subject; and

[0159] (c) contacting the cardiac cells with the cardioprotective agent in combination with the anti -cancer agent reduces the level of cardiotoxicity for the anti-cancer agent in the cardiac cells (e.g., the further level of cardiotoxicity is less than the level of cardiotoxicity described herein). In such instances, the anti-cancer agent may be administered in combination with the cardioprotective agent at a standard or conventional dose. For other examples, the anti -cancer agent is administered in combination with the cardioprotective agent at a high dose. In alternative examples, the anticancer agent is administered in combination with the cardioprotective agent at a low dose.

[0160] The terms, “lower”, “reduced” and “decreased”, as used herein refer to a lower level of cardiotoxicity in the presence of the cardioprotective agent and the anti-cancer agent, when compared to a level of cardiotoxicity measured in the presence of the anti-cancer agent in isolation (i.e., with no cardioprotective agent). The level of cardiotoxicity or therapeutic efficacy may be relative or absolute (i.e., relatively or absolutely lower, reduced or decreased). In some examples, the level of cardiotoxicity of the anti-cancer agent is lower, reduced or decreased in the presence of the cardioprotective agent if its level is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or 0.0001% of the level of cardiotoxicity observed in the subject’s cardiac cells when contacted with the anti-cancer agent and in the absence of the cardioprotective agent.

[0161] In another form, the present disclosure provides a method of determining or predicting a likelihood of a cardioprotective agent to prevent or inhibit cardiotoxicity of an anti-cancer agent in a subject, said method including the steps of:

[0162] (a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject (e.g., contacting or culturing the cardiac cells with the anti-cancer agent);

[0163] (b) determining a further level of cardiotoxicity for the anti-cancer agent and the cardioprotective agent in the cardiac cells of the subject (e.g., contacting or culturing the cardiac cells, such as sequentially or simultaneously, with the anti-cancer agent and the cardioprotective agent); and

[0164] (c) determining the likelihood of the cardioprotective agent to prevent or inhibit cardiotoxicity of the anti-cancer agent in the subject based on the determinations made in steps (a) and (b).

[0165] The present methods may further include the step of determining the level of therapeutic efficacy for the anti-cancer agent in combination with the cardioprotective agent, such as by those methods provided herein.

[0166] It is envisaged that such a method may further include the step of administering the anti- cancer agent and the cardioprotective agent to the subject when the determination in step (c) indicates a medium or high likelihood of the cardioprotective agent at least partly preventing or inhibiting cardiotoxicity of the anti -cancer agent in the subject.

[0167] As used herein, the term “cardioprotective agent” refers to any compound or molecule (or to any mixture of compounds) that protects the subject’s heart and associated vasculature from a toxic substance (e.g., an anti-cancer agent), such as by stabilizing the cellular membrane of a cardiac cell, normalizing cardiac cellular functions, reducing oxidative stress or reactive oxygen species production and / or reducing inflammation.

[0168] Suitably, the anti-cancer agent described herein may be co-administered with (simultaneously or sequentially) a cardioprotective agent for the treatment or prevention of drug- induced cardiotoxicity, such as that described herein. Non-limiting examples of such cardioprotective agents include a SGLT2 inhibitor (e.g., canagliflozin, dapagliflozin, and empagliflozin), an aldosterone antagonist (i.e., a mineralocorticoid receptor antagonist, such as spironolactone and eplerenone), an aldosterone synthase inhibitor (e.g., lorundrostat), a thiazide diuretic (e.g., chlorothiazine, hydrochlorothiazide, metolazone), a beta blocker (e.g., a beta- adrenergic blocking agent, such as acebutolol, atenolol, bisoprolol, carvedilol, metoprolol, nadolol, nebivolol, penbutolol, propranolol), an angiotensin-convertine enzyme (ACE) inhibitor (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolarpil), an angiotensin II receptor blocker (e.g., ARBs, such as candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan), a calcium channel blocker (e.g., amlodipine, diltiazem, felodipine, isradipine, nicardipine, nifedipine, nisoldipine, verapramil), a renin inhibitor (e.g., Aliskiren), an alpha blocker (e.g., an alpha-adrenergic antagonist, an alpha-adrenergic blocking agent, an adrenergic blocking agent, an alpha-blocking agent, such as doxazosin, prazosin, terazosin, tamsulosin, alfuzosin), an alpha-beta blocker (e.g., an alpha-beta adrenergic blocker, such as carvedilol, labetalol), a central -acting agent (e.g., a central adrenergic inhibitor, a central alpha agonist, a central agonist, such as clonidine, guanfacine, methyldopa), a vasodilator (e.g., hydralazine, minoxidil), an endothelin antagonist (e.g., an endothelin receptor antagonist, such as macitentan, bosentan, darusentan, sitaxsentan, tezosentan, ambrisentan, atrasentan, avosentan, clazosentan, zibotentan, edonentan, enrasentan, danusentan), a phosphodiesterase (PDE) inhibitor (e.g., a phosphodiesterase V inhibitor, such as tadalafil, sildenafil and vardenafil), an endopeptidase inhibitor, a lipid lowering agent (e.g., HMG CoA reductase inhibitors or statin such as simvastatin, pravastatin, atorvastatin, lovastatin, itavastatin, fluvastatin, pitavastatin, rosuvastatin, ZD-4522 and cerivastatin; fibrates or PPARa activators, such as clofibrate, fenofibrate and gemfibrozil), an angiotensin receptor neprilysin inhibitor (ARNI; e.g., sacubitril -valsartan), an ACE neprolysin inhibitor (e.g., omapatrilat), a guanylate cyclase stimulator (e.g., vericiguat), a cardiac-specific myosin activator (e.g., omacamtive mecarbil), an antioxidant (e.g., vitamins A, E, and C, P-carotene, taurine, apocynin and zinc), an iron-chelating agent (e.g., dexrazoxane), a late inward sodium current (INaL) inhibitor (e.g., ranolazine), a metabolic agent (e.g., butyric acid, beta-hydroxybutyrate), a growth factor (e.g., neuregulin-1, granulocyte colony-stimulating factor, erythropoietin) and a HD AC inhibitor (e.g., givinostat).

[0169] In light of the present disclosure, the person skilled in the art may conduct screening assays to identify, design or produce an agent, or more particularly a cardioprotective agent, for use in preventing cardiotoxicity from an anti-cancer agent in a subject having cancer.

[0170] As such, in a related form there is provided a method of identifying, designing or producing a cardioprotective agent for use in inhibiting or preventing cardiotoxicity from an anti-cancer agent in a subject having cancer, said method including the steps of:

[0171] (a) determining a first level of cardiotoxicity for the anti-cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject; (b) determining a second level of cardiotoxicity for the anti-cancer agent and a candidate agent in the cardiac cells;

[0172] (c) based on the determinations in (a) and (b), determine whether the candidate agent inhibits or prevents cardiotoxicity of the anti -cancer agent in the subject.

[0173] Such a method may further include contacting the cardiac cells with the anti -cancer agent to determine the first level of cardiotoxicity and contacting the cardiac cells with the anti -cancer agent and the candidate agent to determine the second level of cardiotoxicity.

[0174] Cell culture

[0175] Suitably, determining a level of cardiotoxicity and / or a level of therapeutic efficacy comprises contacting the respective cell types (i.e., cardiac cells and cancer cells) with the anticancer agent (and with or without a cardioprotective agent), such as for a time and under conditions suitable to determine such levels. In such examples, contacting these cells generally includes culturing the cells in the presence or absence of the anti-cancer agent. In some examples, the cells are cultured in a culture medium under suitable culture conditions in the presence or absence of the anti-cancer agent.

[0176] A “culture medium” (also referred to herein as a “cell culture medium” or “medium”) is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation. The present disclosure contemplates various parameters and conditions for culturing the cells described herein. The cell culture medium may contain any of the following nutrients in appropriate amounts and combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc, as required for the cell type in question. In this regard, it is contemplated that different cell culture media may be used for each of the cardiac cells and cancer cells of the subject.

[0177] Cell culture media are known in the art and may be classified as natural or artificial media. Examples of cell culture media include but are not limited to Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute Medium (RPMI). Selection of an appropriate medium for culturing the cell is within the capability of the skilled artisan. In particular examples, the cell culture medium comprises a cardiac spheroid culture media. Suitably, the culture medium is supplemented with a glutamine or derivative thereof, such as L-glutamine or GlutaMAX™ Supplement (i.e., L-alanyl-L-glutamine dipeptide in 0.85% NaCl). Typically, the culture media is supplemented with a concentration range of a glutamine or glutamine derivative of between about 0.1 mM to about 2 mM, and even more particularly between about 0.25 mM and about 1 mM. In alternative examples, the culture media, such as that for culturing cardiac spheroids, contains no or substantially no (e.g., less than 0.05, 0.01, 0.005 or 0.001 mM) glutamine, such as L-glutamine. For some examples, the culture media, such as that for culturing cardiac spheroids, contains no or substantially no (e.g., less than 0.05, 0.01, 0.005 or 0.001 mM) antibiotic, such as penicillin and / or streptomycin.

[0178] Suitable conditions include growing the cell under standard cell culture conditions in a cell culture incubator (e.g., at 37°C. in a humidified atmosphere of >80% relative humidity air and 5 to 10% CO2). Typically, the culturing is performed at a temperature between about 25°C and about 40°C. More specifically, the culturing is generally performed at a temperature between about 35°C and about 38°C. Even more specifically, culturing is performed at a temperature of between about 36.5°C and about 37.5°C. By way of an illustrative example, culturing may suitably be performed at about 37°C. Furthermore, culturing typically occurs at between about 2% CO2 and 10% CO2. In some particularly preferred embodiments, culturing occurs at about 5% CO2. During culturing, the culture media may be supplemented by one or more antibiotics such as ampicillin or penicillin as required.

[0179] According to particular examples, the cells (i.e., the cardiac cells and / or cancer cells) are contacted with the anti-cancer agent and optionally the cardioprotective agent (e.g., cultured in the presence of the anti-cancer agent with or without the cardioprotective agent) for at least about 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or any range therein. In some examples, the cells are contacted with the anti-cancer agent (e.g., cultured in the presence of the anti-cancer agent) and optionally the cardioprotective agent, for 48 hours or less, 36 hours or less, 24 hours or less, or 12 hours or less.

[0180] In various examples, the cells (i.e., the cardiac cells and / or cancer cells) are contacted or cultured with the anti-cancer agent and optionally the cardioprotective agent (e.g., cultured in the presence of the anti-cancer agent with or without the cardioprotective agent) for between about 30 minutes to about 14 days. More particularly, the cells are contacted or cultured with the anti-cancer agent and optionally the cardioprotective agent for between about 2 hours and about 7 days, between about 2 hours and about 5 days, between about 6 hours and about 7 days, between about 6 hours and about 72 hours, between about 6 hours and about 48 hours, between about 6 hours and about 24 hours, between about 12 hours and about 7 days, between about 12 hours and about 72 hours, between about 12 hours and about 48 hours and between about 12 hours and about 24 hours.

[0181] For various examples, the cells (i.e., the cardiac cells and / or cancer cells) are contacted with the cardioprotective agent for at least about 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or any range therein, before being contacted with the anti-cancer agent. In alternative examples, the cells (i.e., the cardiac cells and / or cancer cells) are contacted with the anti-cancer agent for at least about 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or any range therein, before being contacted with the cardioprotective agent.

[0182] Suitably, the culture medium is supplemented with a mammalian serum, such as foetal bovine serum (FBS). Typically, the culture media is supplemented with a mammalian serum at a concentration of between about 1% v / v to about 20% v / v (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% v / v or any range therein), about 5% v / v to about 15% v / v, about 7.5% v / v to about 12.5% v / v or more particularly about 10% v / v. In particular examples, the cells described herein, (i.e., the cardiac cells and / or cancer cells) are cultured in a medium comprising the subject’s blood, serum or plasma in an attempt to replicate in vivo physiological conditions (e.g., circulating hormones, inflammasome mediators, inflammatory cytokines, biomarkers) specific to the subject in culture.

[0183] Cardiac cells

[0184] As used herein the term “cardiac cell” refers to any cell present in the heart that provides a cardiac function, such as heart contraction or blood supply, or otherwise serves to maintain the structure and / or function of the heart. To this end, cardiac cells may be any cell that constitute the cardiac muscle, blood vessels and cardiac cell supporting structure of the heart. It is also envisaged that the term encompass cells that exist in the epicardium, myocardium or endocardium of the heart. Cardiac cells can include, for example, cardiac muscle cells or cardiomyocytes, and cells of the cardiac vasculatures, such as cells of a coronary artery or vein. Other non-limiting examples of cardiac cells include cardiac epithelial cells, cardiac endothelial cells, cardiac fibroblasts, cardiac smooth muscle cells, cardiac leucocytes, cardiac pericytes, epicardial cells, cardiac stem or progenitor cells, cardiac conducting cells, cardiac nerve cells and cardiac pacemaking cells. Cardiac cells may be derived from stem cells, including, for example, embryonic stem cells or induced pluripotent stem cells. In particular examples, the cardiac cell is or comprises a cardiomyocyte.

[0185] It is envisaged that the methods described herein may be performed in relation to cells in vitro or ex vivo. In some examples, the method is performed in vitro, such as with cardiac cells isolated from a subject or cardiac organoid or spheroid culture, as described herein.

[0186] Suitably, the cardiac cells described herein are derived, produced or generated from blood- derived cells, and more particularly blood-derived stem cells, obtained or derived from the subject in question. The terms “blood cells”, “population of blood cells”, “blood-derived cells” or “population of blood-derived cells” and the like, as used herein refer to cells, cell populations or cell groups which may be present in the bloodstream in vivo, but not necessarily limited to peripheral blood cells. In this regard, the blood-derived cells may also be found or be able to be isolated from a cell population from, for example, bone marrow or umbilical cord blood, as well as pleural, peritoneal, cerebrospinal or synovial fluids or from various tissues, such as spleen and lymph node. In specific embodiments, the blood- derived cells express one or more marker molecules of blood cells, such as CD45 and / or CDl la.

[0187] According to some examples, the blood-derived cells are or comprise blood-derived mononuclear cells, such as peripheral blood mononuclear cells (PBMCs). The terms “peripheral blood mononuclear cell” or “PBMC” relate to a peripheral blood cell having a round nucleus. These cells typically include lymphocytes (T cells, B cells, NK cells) and monocytes, whereas erythrocytes and platelets have no nuclei, and granulocytes (neutrophils, basophils, and eosinophils) have multi-lobed nuclei. These cells can be extracted from whole blood using Ficoll and gradient centrifugation, which will separate the blood into a top layer of plasma, followed by a layer of PBMCs and a bottom fraction of polymorphonuclear cells (such as neutrophils and eosinophils) and erythrocytes, as hereinafter described.

[0188] Therefore, the present methods can further include the initial step of isolating the blood- derived cells from the subject. Specifically, in some examples the methods include the step of isolating PBMCs from the subject. This may be achieved by any method known in the art, such as Ficoll medium and gradient centrifugation, fluorescence activated cell sorting (FACS) and magnetic beads cell sorting (MACS). Suitably, PBMCs are positively selected from a heterogenous population of cells by the use of one or more cell surface markers, such as CD3, CD4, CD8, CD19, CD20, CD56 / 16, CD14, CD25, CD69, Granzyme B, Perforin, FOXP3, and Helios. Other methods can include the isolation of PBMCs by depletion of non-PBMCs (negative selection).

[0189] The methods described herein may also include the initial or earlier step of producing or generating cardiac cells from the blood-derived cells of the subject. In certain examples, the cardiac cell is derived from maturation of progenitor cells, embryonic stem cells (e.g., human embryonic stem cells) and / or induced pluripotent stem cells (iPSCs), such as into cardiomyocytes, cardiac fibroblasts and / or endothelial cells. In other examples, the cardiac cell is contained within or otherwise associated with an Engineered Heart Tissue (EHT), a cardiac spheroid or a cardiac organoid. Methods of cardiomyocyte maturation from progenitor cells, embryonic stem cells and iPSCs and the generation of cardiac organoids are known in the art, such as described in PCT / AU2017 / 050905; Sahara et al., 2015, EMBO J, Vol.34:710-738; Kattman et al., 2011, Cell Stem Cell, Vol.8:228-240; Burridge et al., Nat Methods, Vol. 11 :855-860, which are incorporated by reference herein.

[0190] A “progenitor cell” is a cell which is capable of differentiating along one or a plurality of developmental pathways, with or without self-renewal. Typically, progenitor cells are unipotent or oligopotent and are capable of at least limited self-renewal.

[0191] The term “embryonic stem cell” refers to cells derived, obtainable or originating from embryos or blastocysts, which are self-renewing and pluri- or toti-potent, having the ability to yield all of the cell types present in a mature animal. Human embryonic stem cells (hESCs) can be isolated, for example, from human blastocysts obtained or derived from human in vivo preimplantation embryos, in vitro fertilized embryos, or one-cell human embryos expanded to the blastocyst stage.

[0192] The terms “induced pluripotent stem cell” and “iPSC” refer to cells derivable, obtainable or originating from human adult somatic cells of any type reprogrammed to a pluripotent state through the expression of exogenous genes, such as transcription factors, including OCT4, SOX- 1, 2, 3, 15 and 18, KLF4, LIN28, Glis 1 and c-MYC, although without limitation thereto.

[0193] Suitably, the blood-derived cells, or more particularly the PBMCs, described herein are reprogrammed or converted to stem cells, such as an induced pluripotent stem cells (iPSCs), capable of generating or being converted into cardiac cells, such as those described herein. As such, the present methods may include the initial or earlier step of generating cardiac cells from the blood-derived cells of the subject, such as by generating stem cells from the blood-derived cells and then subsequently generating cardiac cells from the stem cells. A number of protocols known in the art have been developed to reprogram blood-derived cells, such as PBMCs, into expandable stem cell, or more particularly iPSC, populations, and to derive potentially functional cardiac cells in a controlled manner, such as provided in Example 1.

[0194] It is contemplated that the cardiac cells described herein may be cultured by any means or method known in the art, such as, but not limited to, a 2D culture, a 2D co-culture (e.g., cardiac cells with endothelial cells and / or interstitial cells), a 2.5D culture (e.g., cardiac cells growing on top of a layer of an extracellular matrix (ECM) protein or a hydrogel) or a 3D culture (e.g., a cardiac organoid or spheroid). Referring to certain examples, the cardiac cells are cultured, at least in part, in a 3D culture system.

[0195] Suitably, the cardiac cells are cultured as a cardiac organoid. In alternative examples, the cardiac cells are cultured as a cardiac spheroid. The term “cardiac organoid” refers to an artificial three-dimensional structure comprising cells occurring in heart tissue. Methods of generating cardiac organoids are well known in the art and typically involve several culture steps as well as the use of a matrix and defined factors (see, e.g., Lee et al., 2020, Nat. Commun., Vol. 11 :4283; Shkumatov et al., 2014, PLoS ONE, Vol. 9; Hoang et al., 2018, Nat Protocol, Vol. 13 : 723-737; Drakhlis et al., 2021, Nat. Biotechnol., Vol.39:737-746; Lewis-Israeli et al., 2021, Nat. Commun., Vol. 12:5142).

[0196] Similarly, the term “cardiac spheroid” refers to a three-dimensional cell cluster including a number of aggregated cells that occur in heart tissue. Spheroids are typically simple clusters of cells that do not require a scaffold or substrate to form 3D cultures, rather they do so by simply aggregating together. Unlike organoids, however, spheroids generally do not self-assemble or regenerate and, thus, aren't as advanced as organoids. Conversely, organoids are typically complex clusters of organ-specific cells that can self-assemble when provided a scaffold or substrate that mimics the extracellular environment. Methods of generating cardiac spheroids are also well known in the art, such as those described in Sharma et al., STAR Protocols, Volume 3, Issue 4, (2022); and Sharma & Gentile, J. Vis. Exp. (167), e61962, (2021), which are incorporated by reference herein. Notwithstanding the above, both cardiac organoids and cardiac spheroids suitably mimic the physiological, biochemical and / or molecular responses or reactions of native heart tissue to, for example, therapeutic agents, such as the anti-cancer agent described herein.

[0197] Referring to particular examples, the cardiac cells, inclusive of cardiac organoids and cardiac spheroids, comprise one or more cell types selected from the group consisting of cardiomyocytes, vascular cells (e.g., endothelial cells, smooth muscle cells, pericytes), interstitial cells (e.g., fibroblasts) and neural cells (e.g., neurons / sino-atrial node cells). More particularly, the cardiac cells, the cardiac organoid or the cardiac spheroid suitably comprises, consists of or consists essentially of cardiomyocytes, interstitial cells (e g., fibroblasts) and vascular cells (e g., endothelial cells). Even more particularly, the cardiac cells, the cardiac organoid or the cardiac spheroid suitably comprises, consists of or consists essentially of cardiomyocytes, fibroblasts and endothelial cells.

[0198] Screening methods

[0199] Also described herein are methods for determining the therapeutic responsiveness of a subject’s cancer to an anti -cancer agent. The therapeutic responsiveness determined for the subject may then be utilised as an inclusion criterion and / or to stratify cancer subjects in a clinical trial setting. Suitably, such a method includes the steps of: (a) determining a level of cardiotoxicity for the anti-cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject; and (b) determining a level of therapeutic efficacy for the anti -cancer agent in the subject’s cancer. The determinations made in steps (a) and (b) can indicate or correlate to the therapeutic responsiveness of the subject’s cancer to the anti-cancer agent. Particular broad examples, include the step of treating the subject following predicting a positive responsiveness of the subject’s cancer to the anti-cancer agent (i.e., the anti-cancer agent is administered to the subject when (a) the level of therapeutic efficacy indicates or correlates with relatively increased responsiveness of the cancer to the anti-cancer agent; and optionally (b) the level of cardiotoxicity indicates or correlates with an intermediate or low likelihood of cardiotoxicity from administration of the anti-cancer agent). Accordingly, these embodiments relate to using information obtained about the predicted responsiveness of the subject’s cancer to the anti-cancer agent to thereby construct and implement a treatment regime for the subject. Advantageously, this is personalized to a particular patient so that the treatment regime is optimized for that particular patient.

[0200] Accordingly, in one form, the present disclosure provides a method of stratifying a subject having cancer for a clinical trial of an anti-cancer agent, said method including the steps of:

[0201] (a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject;

[0202] (b) determining a level of therapeutic efficacy for the anti-cancer agent in the subject’s cancer; and

[0203] (c) stratifying the subject for the clinical trial based on the results of determining steps (a) and (b).

[0204] In another related form, the present disclosure provides method of determining inclusion of a subject having cancer in a clinical trial of an anti-cancer agent, said method including the steps of:

[0205] (a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject;

[0206] (b) determining a level of therapeutic efficacy for the anti -cancer agent in the subject’s cancer; and

[0207] (c) determining whether the subject having cancer is included in the clinical trial based on determining steps (a) and (b).

[0208] In the case of prospective trials, in vitro determination of the levels of therapeutic efficacy and cardiotoxicity of prospective patients may be used to stratify patients prior to their entry or inclusion into the trial or while they are enrolled in the trial. In clinical research, stratification is the process or result of describing or separating a patient population into more homogeneous subpopulations according to specified criteria. Stratifying patients initially rather than after the completion of a trial is frequently preferred, for example, by regulatory agencies such as the U.S. Food and Drug Administration that may be involved in the approval process for a therapeutic agent. In some cases, patient stratification may be required by the study design. It will be appreciated that further stratification criteria may be employed in conjunction with determining levels of therapeutic efficacy and cardiotoxicity. Commonly used criteria include age, family history, cancer type, stage and / or grade, etc. Stratification is frequently useful in performing statistical analysis of the results of a trial.

[0209] Kits

[0210] The present disclosure further provides a kit for predicting the responsiveness of a cancer in a subject to an anti-cancer agent, the kit comprising: (a) a first reagent capable of determining a level of cardiotoxicity for the anti-cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject, wherein the level of cardiotoxicity indicates or correlates with a relatively increased or decreased risk of cardiotoxicity in the subject with administration of the anti-cancer agent; and (b) a second reagent capable of determining a level of therapeutic efficacy for the anti-cancer agent in the subject’s cancer, wherein the level of therapeutic efficacy indicates or correlates with relatively increased or decreased responsiveness of the subject’s cancer to the anti-cancer agent.

[0211] In certain examples, the kit further comprises reference data for correlating: (a) the level of therapeutic efficacy with responsiveness of the subject’s cancer to the anti-cancer agent and / or (b) the level of cardiotoxicity with the likelihood or risk of cardiotoxicity in the subject with administration of the anti-cancer agent.

[0212] In particular examples, the reference data is on a computer-readable medium (e.g., software embodying or utilised by any one or more of the methodologies or functions described herein). The computer-readable medium can be included on a storage device, such as a computer memory (e.g., hard disk drives or solid-state drives) and suitably comprises computer readable code components that when selectively executed by a processor implements one or more methods of the present disclosure.

[0213] Suitably, the present kit is for use in any of the methods described herein.

[0214] In a particular embodiment, the present kit provides a “companion diagnostic” whereby information with respect to the levels of therapeutic efficacy and cardiotoxicity are utilized by a clinician or similar for the safe and effective administration of an anti-cancer agent.

[0215] So that preferred embodiments of the present disclosure may be fully understood and put into practical effect, reference is made to the following non-limiting examples. Examples

[0216] Example 1.

[0217] This prophetic Example provides an embodiment of the methods provided herein by the present disclosure.

[0218] A. Determining a level of cardiotoxicity

[0219] (i) Cardiac spheroid production

[0220] A schematic illustration of the production of cardiac spheroids by the present Example is provided in Figure 1.

[0221] Peripheral blood mononuclear cells (PBMCs) isolation from patients and expanded to erythroid progenitor cells (EPCs)

[0222] Peripheral blood was collected from a cancer patient (40 mL in heparin tubes). Patients’ blood was topped with 2% FBS in PBS (1 : 1) and added in SepMate™-50 tube with Lymphoprep at the bottom of the tube (Stemcell Technologies, Vancouver, Canada). The pre-enriched cells were isolated by spinning down at 1200g x lOmin at room temperature. The top layer solution containing the pre-enriched cells was collected and diluted with 2% FBS in PBS, then centrifuged at 300g x 8 min. PBMCs were resuspended and expanded using Erythroid Progenitor Reprogramming Kit (Stemcell Technologies, Vancouver, Canada) following the manufacturer’s instructions.

[0223] Reprogramming erythroid progenitor cells (EPCs) to induced pluripotent stem cells (IPSC) and IPSCs cryopreservation

[0224] EPCs were expanded for 7 days, then collected and centrifuged at 300g x 5min (approximately lx 106cells). lOOpl of Human CD34+Cell Nucleofector™ Kit (Lonza, Basel, Switzerland) and 5 pl of Epi5™ Episomal iPSC Reprogramming Kit (Thermofisher, Massachusetts, USA) were added to the cell pellet and transfected using the Lonza 4D- Nucleofector device (CD34+cells program). The cells were then plated in Erythroid expansion medium (Stemcell Technologies, Vancouver, Canada) in a 6-well plate, which was coated overnight with Vitronectin XF (Stemcell Technologies, Vancouver, Canada) following the manufacturer's guidelines. Starting from day 3, cells were cultured in ReproTeSR™ Medium (Stemcell Technologies, Vancouver, Canada) and IPSCs colonies started to form at day 11. IPSCs were characterised by phase-contrast microscopy, Olympus (Tokyo, Japan) and were passaged manually using the microscope and 21G needles to cut in small pieces the IPSCs’ colonies and / or using ReLeSR (Stemcell Technologies, Vancouver, Canada) following the manufacturer's guidelines. 1 x 106IPSCs were collected and spun down at 300g x 5min and cryopreserved in 1ml CryoStor® CS2 (Stemcell Technologies, Vancouver, Canada).

[0225] Generation of IPSC-cardiomyocytes (IPSC-CMs) and IP SC-endothelial cells (IPSC-ECs)

[0226] The cryopreserved IPSC are plated in a 6 well plated coated overnight with Coming® Matrigel® hESC-qualified Matrix (Coming, New York, USA) for 2 weeks / 2 passages before starting IPSCs differentiation to IPSCs-cardiomyocytes (IPSCs-CMs) and IPSCs-endothelial cells (IPSCs-ECs).

[0227] Following the manufacturer’s guidelines, IPSC were plated in 12 well plate coated overnight with Matrigel and differentiated for 14 days with the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit (Stemcell Technologies, Vancouver, Canada). IPSC-CMs contractility was observed using EVOS™ M7000 Imaging System (Thermofisher, Massachusetts, USA). At day 16, IPSC-CMs were dissociated by STEMdiff™ Cardiomyocyte Dissociation Kit (Stemcell Technologies, Vancouver, Canada). The cells are then added to EasySep™ Human PSC- Derived Cardiomyocyte Enrichment Kit which isolate human PSC-derived cardiomyocytes from unwanted cells with tetrameric antibody complexes. The labelled cells are therefore separated using EasySep™ magnet.

[0228] For the generation of IPSC-ECs, IPSC were plated in 6 well plate coated overnight with Matrigel and differentiated by using STEMdiff™ Endothelial Differentiation Kit (Stemcell Technologies, Vancouver, Canada) and expanded with STEMdiff™ Endothelial Expansion Medium Kit (Stemcell Technologies, Vancouver, Canada).

[0229] For both IPSC-CMs and IPSC-ECs, around 15,000 cells were re-plated to be fixed and immunolabelled while 1,215,000 IPSC-CMs and 607,500 IPSC-ECs were used for cardiac spheroids (CSs) formation. The remaining IPSC-CMs and IPSC-ECs were cryopreserved in 1ml STEMdiff™ Cardiomyocyte Freezing Medium (Stemcell Technologies, Vancouver, Canada) and 1ml Crystor, respectively.

[0230] Cell culture Human Cardiac Fibroblast (HCF) (Sigma-Aldrich, Missouri, USA) were cultured in Cardiac Fibroblast Growth Media (Sigma-Aldrich, Missouri, USA) both supplemented with 1% penicillin streptomycin (Thermofisher, Massachusetts, USA) and 1% L-glutamine (Thermofisher, Massachusetts, USA). Media was changed every 2-3 days and cells were cultured until 70-90% confluency before being passaged with trypsin-EDTA solution (Sigma-Aldrich, Missouri, USA). Human coronary artery endothelial cells (Cell Applications, Inc., San Diego, CA, USA) were cultured in MesoEndo Cell Growth Medium (Cell Applications, Inc., San Diego, CA, USA). Human induced pluripotent stem cell-derived cardiomyocytes were cultured on fibronectin (bovine, Sigma-Aldrich, Darmstadt, Germany) pre-coated flasks using iCell Cardiomyocytes Plating Medium (Fujifilm Cellular Dynamics, Inc., Madison, Wisconsin, USA). Plating Medium was replaced with iCell Cardiomyocytes Maintenance Medium (Fujifilm Cellular Dynamics, Inc., Madison, Wisconsin, USA).

[0231] Cardiac spheroid (CS) formation

[0232] Prior to cardiac spheroid formation, agarose powder (Sigma-Aldrich, Missouri, USA) and 3D micromoulds (Sigma-Aldrich, Missouri, USA) were sterilised. Agarose powder (1%) was reconstituted in phosphate buffered saline (PBS) solution, then heated for a few seconds to allow proper dissolution of the powder, and finally pipetted into 3D micromoulds to set and form moulds for spheroids. Solidified moulds were submerged in culture media and incubated at 37°C and 5% CO2, following manufacturer’s guidelines.

[0233] CSs were formed according to the previously published procedure (Sharma et al., STAR Protocols, 2022). HCFs, IPSC-CMs and IPSC-ECs described above were detached from their respective flasks and counted with trypan blue (Thermofisher, Massachusetts, USA) and haemocytometer (Sigma- Aldrich, Missouri, USA). They were then mixed in a ratio of 2: 1 : 1 (IPSC- CMs: IPSC-ECs: HCF) and centrifuged at 300g x 5min. Cell pellet was resuspended in cardiac spheroid culture media (IPSC-CMs: IPSC-ECs: HCF media free from penicillin streptomycin and L-glutamine at a ratio of 2: 1 : 1, respectively). Cell suspension ( 190 pl) was added to the inner cavity of the agarose moulds and incubated at 37°C and 5% CO2 with daily media changes. Following 48 hours, CS were collected and carefully transferred to a 96-well plate (a minimum of five CS per well). Media was replaced with a solution containing 1 : 1 collagen 1 rat tail (Merck, Massachusetts, USA) and the whole plate was moved to an incubator at 37°C and 5% CO2 overnight.

[0234] (ii) Tumoroid production Patient-specific cancer cells were isolated from a biopsy of the patient’s cancer and processed to generate a cell suspension thereof. Tumoroid production was based on a bioprinting platform. A matrix, containing fibronectin, collagen type IV, and laminin was utilised.

[0235] All reagents and cartridges were purchased commercially, such as from Inventia Life Sciences. The appropriate bioinks, together with an activator, sterile filtered 70% ethanol, and sterile filtered milli-Q water was added to the cartridge. The cartridge containing the reagents and a 96-well plate were placed inside the bioprinter to print the base matrix. Next, IxlO6cells was suspended in 200 pL of the activator and placed in the cartridge to print cells. Once printing of the cells was completed, the 96-well plate was topped up with culture media. Tumoroids were then allowed to grow and develop for a total of 7 days prior to the addition of an anti -cancer agent.

[0236] (iii) Determining therapeutic efficacy and potential cardiotoxicity

[0237] Each anti-cancer agent studied was added into wells containing the cardiac spheroids at a series of concentrations (e.g., 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500 and 1000 pM) in triplicate so as to generate a dose response curve. The same concentrations of the anti-cancer agent were also added into wells containing the tumoroids in triplicate. Appropriate no drug controls or vehicle controls were also added in triplicate to the wells of both the cardiac spheroids and the tumoroids. Plates were returned to the incubator for a further 72 h before assessing levels of cardiotoxicity and therapeutic efficacy.

[0238] Cell viability for both the cardiac spheroids and the tumoroids was assessed by an MTSbased method (e.g., CellTiter 96® AQueous One Solution Cell Proliferation Assay). 20 pL of an MTS-containing solution reagent was directly added to each well, plates were covered with aluminium foil, and were placed in the incubator for three hours. The absorbance was read on a spectrophotometer at a wavelength of 490 nm.

[0239] All raw absorbance values were corrected for background absorbance by subtraction of the mean blank (no MTS) absorbance value and the triplicate readings of absorbance were used to calculate a mean absorbance. The percentage viability was determined using the formula below.

[0240] Mean absorbance (drug concentration)

[0241] % viability = — - -; ;- - - - - - — x 100

[0242] Mean absorbance (no drug control)

[0243] Three biological replicate trials were conducted for each treatment group. Dose-response curves were then generated for each of the anti-cancer agents studied. Data was analysed using non-linear regression; [Inhibitor] vs. response and were fit to four parameter model (Y = Bottom + (Top - Bottom) / (1 + (IC50 / X)ASlope). Half maximal inhibitory concentration for each compound was calculated. Variation among the biological repeats was presented as a standard error of mean (SEM). Differences in IC50 values were assessed by ordinary one-way Analysis of variance (ANNOVA) tests.

[0244] Example 2.

[0245] This Example provides an embodiment of the methods provided herein by the present disclosure. More particularly, the present Example was designed to evaluate a patient-specific responses to the cardiotoxic chemotherapeutic agent, doxorubicin, in cardiac spheroids that were generated from PBMCs from seven different patients.

[0246] Materials and Methods

[0247] Drugs

[0248] Doxorubicin hydrochloride (used at 1, 5, 10, 20, and 40 pM) was purchased from Sigma- Aldrich (USA).

[0249] Generation of iPSCs from patient-derived blood

[0250] Peripheral blood was collected in EDTA-coated tubes, diluted with 2% FBS in PBS (1 : 1), and added to SepMate™-50 tubes containing Lymphoprep™ density gradient medium (STEMCELL Technologies, Vancouver, Canada). Tubes were centrifuged at 1200*g for 10 minutes at room temperature to collect peripheral blood mononuclear cells (PBMCs) in the top layer solution. This was diluted with 2% FBS in PBS, centrifuged at 300g x 8 min, and resuspended in an Erythroid Progenitor Reprogramming Kit (STEMCELL Technologies, Vancouver, Canada), following the manufacturer’s instructions. At seven days in this expansion media, approximately 1 x io6cells were collected and centrifuged at 300 g for 5 minutes. The cell pellet was resuspended in 100 pL of Human CD34+Cell Nucleofector™ Solution (Lonza, Switzerland) containing 5 pL of the Epi5™ Episomal iPSC Reprogramming Kit (Thermo Fisher Scientific, USA). Cells were transfected using the Lonza 4D-Nucleofector™ system with the CD34+cellspecific program. Following transfection, cells were plated in erythroid expansion medium (STEMCELL Technologies, Canada) in 6-well plates previously coated overnight with Vitronectin XF™ (STEMCELL Technologies, Canada), following the manufacturer’s instructions. Beginning on day 10, the culture medium was switched to ReproTeSR™ (STEMCELL Technologies, Canada) to support reprogramming of EPCs and iPSCs colony formation. Visible iPSC colonies emerged around day 17 and were monitored by phase-contrast microscopy (Olympus, Japan). Colonies were manually passaged using an inverted microscope and 21G needles to segment colony fragments or alternatively dissociated enzymatically using ReLeSR™ (STEMCELL Technologies, Canada).

[0251] Differentiation of iPSCs into Cardiomyocytes (iPSC-CMs) and Endothelial Cells (iPSC-ECs) iPSCs were plated in 6-well plates precoated overnight with Coming® Matrigel® hESC- qualified Matrix (Coming, New York, USA) and cultured for 2 weeks before initiating differentiation into cardiomyocytes (iPSCs-CMs) and endothelial cells (iPSCs-ECs).

[0252] For generating iPSC-CMs, iPSCs were plated in a 12-well plate pre-coated overnight with Matrigel and differentiated over 14 days using the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit (STEMCELL Technologies, Canada). Contractile activity of iPSC-CMs was visually observed with the EVOS™ M7000 Imaging System (Thermofisher, USA). On day 16, the iPSC-CMs were dissociated using the STEMdiff™ Cardiomyocyte Dissociation Kit (STEMCELL Technologies, Canada). The cells were then added to the EasySep™ Human PSC- Derived Cardiomyocyte Enrichment Kit (STEMCELL Technologies, Canada), which isolates iPSC-CMs from unwanted cells with tetrameric antibody complexes. The labeled cells are then separated using the EasySep™ magnet.

[0253] For the generation of iPSC-ECs, iPSCs were plated in a 6-well plate coated overnight with Matrigel and differentiated using STEMdiff™ Endothelial Differentiation Kit (STEMCELL Technologies, Canada). After differentiation, cells were expanded in STEMdiff™ Endothelial Expansion Medium (STEMCELL Technologies) to enrich and stabilize the endothelial population. Only iPSC-ECs at passage 3 or higher were used in this study to ensure phenotypic consistency and reduce contamination by non-endothelial cells.

[0254] Culture of human cardiac fibroblasts

[0255] Human Cardiac Fibroblasts (CFs) (Sigma-Aldrich, USA) were cultured in Cardiac Fibroblast Growth Medium (Sigma-Aldrich, USA) supplemented with 1% penicillin streptomycin (Thermofisher, USA) and 1% L-glutamine (Thermofisher, USA). Culture medium was changed every 2-3 days, and cells were maintained at 70-90% confluency before being passaged with trypsin-EDTA solution.

[0256] Cardiac spheroid formation

[0257] Agarose powder (1%) was reconstituted in PBS solution, briefly heated to ensure full dissolution, and pipetted into sterile 3D micromoulds (Sigma-Aldrich, USA) to form spheroid moulds. Once gellified, the agarose moulds were submerged in culture medium and incubated at 37°C and 5% CO2 to equilibrate.

[0258] Cardiac spheroids (CSs) were generated following protocols established in previous publications from the inventors (see, e.g., Chung Ming et al., 2024, Liu Chung Ming et al., 2025). CFs, IPSC-CMs, and IPSC-ECs were combined at a 2: 1 : 1 ratio (iPSC-CMs : iPSC-ECs : CFs) and centrifuged at 300 * g for 5 minutes. The resulting pellet was resuspended in a mixed cardiac spheroid medium composed of iPSC-CM, iPSC-EC, and CF media at a 2: 1 : 1 ratio, for a total of ~25K cells / CS.

[0259] A 190 pL of the cell suspension was dispensed into the central cavity of each agarose mould and incubated at 37 °C and 5% CO2, with daily media replacement. After four days, CSs were collected and transferred to a 96-well plate, with a minimum of five CSs per well. The culture medium was then replaced with a 4%alginate / 8%gelatin hydrogel and fresh medium. Plates were incubated overnight at 37 °C and 5% CO2 to allow collagen embedding of the spheroids. Drug treatment was conducted after 1 day.

[0260] Toxicity assay

[0261] CSs in alginate / gelatin hydrogels were transferred to 96-well clear bottom black polystyrene microplates (Coming®, USA) 24 hours before their treatment with DOX. Live / Dead® Viability / Cytotoxicity Kit for mammalian cells (Thermofisher, USA) was used according to manufacturer’s instructions to evaluate toxicity ratio, calculated as a ratio between dead versus live cells, measured in each well at 645 nm (for ethidium homodimer) and 530 nm (calcein-AM), respectively. Ratios were normalized against the number of cells present within the spheroid and against the control culture (untreated spheroids). GraphPad Prism™ (La Jolla, CA) was used for data analysis and statistics.

[0262] Results and Conclusions

[0263] A total of seven types (P1-P7) of cardiac spheroids derived from seven different patients were used to test increasing concentrations of doxorubicin between 1 pM and 40 pM (Figure 2). Control (CTL) cardiac spheroids were tested with media only and used as a comparison for any increase in cardiotoxicity. A toxicity ratio (dead / live cells) was measured across all concentrations for each patient. Doxorubicin significantly increases the toxicity ratio already at 10 pM for patients Pl, P4 and P7 (Figures 2A, 2D, and 2G, respectively). For patient P3, doxorubicin significantly increased toxicity at 20 pM (Figure 2C), whereas it was already toxic at lower doses for P5 (5 pM, Figure 2E) and for P6 (1 pM, Figure 2F). In contrast, doxorubicin did not significantly induce any toxicity even at high concentrations for patient P2 (Figure 2B). Altogether, these findings support that patient-derived cardiac spheroids can be used to identify the sensitivity of the heart to cardiotoxic drugs using a personalised approach. This could help to better tailor the drug regimen and potentially prevent the toxicity associated with the use of cancer therapy. Additionally, it will help to test novel protective therapies against the identified toxic concentrations.

[0264] Example 3.

[0265] This prophetic Example provides an embodiment of validation studies for the testing of toxicity and protection of drugs in cardiac spheroids or organoids generated from patient-derived blood. The result of such testing is intended to be compared in this prophetic Example with responses from in vivo preclinical studies in small animals, followed by clinical trials in human patients. This is designed to support the clinical translation of the methods described herein, exemplifying a direct correlation between the toxicity and protection demonstrated in vitro in organoids / spheroids with that seen in patients. Suitably, the present Example is to be performed in breast cancer patients first, before applying it to other common cancers, such as colorectal (CRC), lung (NSCLC), melanoma, ovarian, prostatic cancers, and others.

[0266] Preclinical cardiotoxicity testing

[0267] • Establish an in vivo mouse model of cancer. This model will be used to validate in vitro results in mouse cardiac and cancer spheroids / organoids with in vivo data to test cardiotoxicity risk and cancer response, as described herein. Each mouse will be considered “a patient”, so as to validate in vitro and in vivo results;

[0268] • Establish mouse cardiac and cancer spheroids / organoids from mice mentioned above, which will then be scaled up before commencing production of enough mouse organoids to test multiple drugs.

[0269] Clinical trial translation

[0270] • Start recruiting cancer patients and healthy controls for the proof-of-concept (POC) study, so as to collect blood and generate patient-specific cardiac and cancer spheroids / organoids to test cardiotoxicity risk and cancer response in 30 patients;

[0271] • Start a phase 2 study, which will expand the study to more patients, so to further validate our cardiotoxicity and cancer response results in the POC phase above.

Claims

CLAIMS:

1. A method of predicting the therapeutic efficacy and cardiotoxicity of an anti -cancer agent in a subject having cancer, said method including the steps of:(a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject; and(b) determining a level of therapeutic efficacy for the anti -cancer agent in the subject’s cancer.

2. The method of Claim 1, wherein based on the determinations made in steps (a) and (b), the method further including the step of initiating, continuing, modifying or discontinuing administration of the anti-cancer agent to the subject.

3. The method of Claim 1 or Claim 2, further including the step of administering to the subj ect a therapeutically effective amount of the anti-cancer agent when the level of cardiotoxicity indicates or correlates with a low likelihood of cardiotoxicity in the subject and the level of therapeutic efficacy indicates or correlates with a high likelihood of therapeutic efficacy of the anti-cancer agent in the subject.

4. A method of treating a cancer in a subject, said method including the steps of:(a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject;(b) determining a level of therapeutic efficacy for the anti -cancer agent in the subject’s cancer; and(c) initiating, continuing, modifying or discontinuing administration of the anti-cancer agent to the subject based on the determinations made in steps (a) and (b).

5. The method of any one of the preceding claims, wherein the blood-derived cells are PBMCs.

6. The method of any one of the preceding claims, further including the initial or earlier step of isolating the blood-derived cells from the subject.

7. The method of any one of the preceding claims, further including the step of generating the cardiac cells from the blood-derived cells.

8. The method of any one of the preceding claims, wherein the cardiac cells are cultured, at least in part, in a 3-dimensional culture system.

9. The method of Claim 8, wherein the cardiac cells are or comprise a cardiac organoid or a cardiac spheroid.

10. The method of Claim 9, wherein the cardiac organoid or the cardiac spheroid comprise cardiomyocytes, interstitial cells and vascular cells.

11. The method of any one of the preceding claims, wherein the cardiac cells are cultured in a media comprising blood, plasma or serum derived from the subject.

12. The method of any one of the preceding claims, further including the step of administering to the subject a therapeutically effective amount of a cardioprotective agent in combination with the anti-cancer agent.

13. The method of Claim 12, further including the step of culturing the cardiac cells in the presence of the cardioprotective agent and the anti -cancer agent.

14. The method of Claim 13, further including the step of determining whether contacting the cardiac cells with the cardioprotective agent in combination with the anti-cancer agent modulates the level of cardiotoxicity for the anti -cancer agent in the subject.

15. The method of any one of Claims 12 to 14, wherein the cardioprotective agent is administered in combination with the anti -cancer agent when the level of cardiotoxicity indicates or correlates with a medium or high likelihood of cardiotoxicity in the subject and the level of therapeutic efficacy indicates or correlates with an intermediate or high likelihood of therapeutic efficacy of the anti-cancer agent in the subject.

16. The method of any one of Claims 12 to 15, wherein the cardioprotective agent is administered in combination with the anti -cancer agent when contacting the cardiac cells with thecardioprotective agent in combination with the anti-cancer agent reduces the level of cardiotoxicity for the anti-cancer agent in the cardiac cells.

17. The method of any one of the preceding claims, wherein determining the level of cardiotoxicity comprises contacting the cardiac cells with a dose range of the anti -cancer agent.

18. The method of any one of the preceding claims, wherein the level of cardiotoxicity is determined at least partly by detecting a level or presence of one or more of vascular damage, vascular dysfunction, cardiac cell viability, cardiac cell atrophy, cardiac cell necrosis, cardiac cell apoptosis, cardiac fibrosis, contractile dysfunction, electrophysiological dysfunction, mitochondrial damage, oxidative stress and inflammation.

19. The method of any one of the preceding claims, wherein the anti -cancer agent is selected from the group consisting of: a chemotherapeutic agent, a molecularly targeted agent, a T cell expressing a chimeric antigen receptor, an antibody or antigen-binding fragment thereof, an antibody-drug conjugate, an angiogenesis inhibitor, an immunotherapeutic agent, radiotherapy and any combination thereof.

20. The method of any one of the preceding claims, wherein step (b) includes determining the level of therapeutic efficacy for the anti-cancer agent in cancer cells obtained or derived from the subject’s cancer.

21. The method of Claim 20, wherein the cancer cells comprise a 2D culture, a 2D co-culture, a 2.5D culture, a 3D culture or a patient-derived xenograft.

22. The method of Claim 21, wherein the cancer cells are or comprise a cancer cell line or a patient-derived xenograft.

23. The method of any one of Claims 20 to 22, further including the initial or earlier step of isolating the cancer cells from the subject.

24. The method of any one of Claims 20 to 23, wherein determining the level of therapeutic efficacy comprises contacting the cancer cells with a dose range of the anti-cancer agent.

25. A method of stratifying a subject having cancer for a clinical trial of an anti-cancer agent, said method including the steps of:(a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject;(b) determining a level of therapeutic efficacy for the anti -cancer agent in the subject’s cancer; and(c) stratifying the subject for the clinical trial based on the results of determining steps (a) and (b).

26. A method of determining inclusion of a subject having cancer in a clinical trial of an anticancer agent, said method including the steps of:(a) determining a level of cardiotoxicity for the anti -cancer agent in cardiac cells produced from blood-derived cells obtained or derived from the subject;(b) determining a level of therapeutic efficacy for the anti -cancer agent in the subject’s cancer; and(c) determining whether the subject having cancer is included in the clinical trial based on determining steps (a) and (b).

27. The method of any one of Claims 2 to 28, further including the step of monitoring the subject for cardiotoxicity during administration of the anti -cancer agent when the level of cardiotoxicity indicates or correlates with a medium or high likelihood of cardiotoxicity in the subject.

28. The method of Claim 29, wherein the monitoring step includes detecting a level or presence of one or more of vascular damage, vascular dysfunction, cardiac cell viability, cardiac cell atrophy, cardiac cell necrosis, cardiac cell apoptosis, cardiac fibrosis, contractile dysfunction, calcium signalling and action potential dysfunction, mitochondrial damage, oxidative stress and inflammation in the subject.

29. The method of any one of the preceding claims, further including the step of determining whether contacting the cardiac cells with a cardioprotective agent in combination with the anticancer agent reduces the level of cardiotoxicity for the anti -cancer agent in the subject.

30. The method of any one of the preceding claims, further including the step of determining a further level of cardiotoxicity for the anti -cancer agent in the cardiac cells at a time point following administration of the anti-cancer agent, wherein the cardiac cells are cultured in a media comprising serum derived from the subject at or around the time point.

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