Compositions and methods for treating cardiotoxicity

Abstract

Provided herein are compositions and methods for treating cardiotoxicity in a subject. The compositions and methods provided herein may, for example, be used to characterize the risk of cardiotoxicity in a subject in need of anthracycline treatment. In particular, the treatments provided herein may be applicable to subjects with breast cancer or a hematologic malignancy.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING CARDIOTOXICITY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Patent Application serial number 63 / 631,333, filed April 8, 2024, which is incorporated herein by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. The Sequence Listing .XML file, created on April 2, 2025, is named 167688-020801PCT_SL.xml and is 182,807 bytes in size.

[0006] BACKGROUND OF THE INVENTION

[0007] Anthracyclines are generally effective first line regimens for hematological malignancies and other cancers. However, about 20% of cancer patients administered anthracyclines experience a dose-dependent cardiotoxicity. Although conventional blood biomarkers are often elevated after cytotoxic cancer therapy, they do not serve as a basis for assessing the risk of cardiotoxicity prior to administration of the cancer therapy. Therefore, there is an unmet need for compositions and methods that identify a patient’s risk of developing cardiotoxicity.

[0008] SUMMARY OF THE INVENTION

[0009] The present disclosure provides compositions and methods for identifying a subject at risk of cardiotoxicity and treating or preventing cardiotoxicity in such a subject. In some aspects, the disclosure provides panels for characterizing the risk of cardiotoxicity in a biological sample of a subject in need thereof, e.g., prior to administration of an anthracy cline. In some embodiments, the panels comprise capture molecules that bind specifically to adaptive immune response proteins, wherein the risk of developing cardiotoxicity is associated with an alteration, e.g., an increase, in the level of the adaptive immune response protein.

[0010] In some aspects, the panels disclosed herein comprise capture molecules that bind specifically to a polypeptide biomarker selected from the group consisting of: carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase non-receptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain-containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7. In some embodiments, the panel comprises capture molecules which bind specifically to carboxypeptidase vitellogenic like (CPVL) and polymeric immunoglobulin receptor (PIGR). In some embodiments, the panel comprises capture molecules which bind specifically to carboxypeptidase vitellogenic like (CPVL) and / or polymeric immunoglobulin receptor (PIGR), or a polynucleotide encoding such a biomarker. In some embodiments the capture molecule is an antibody. In some embodiments the capture molecule is an aptamer. In some embodiments the capture molecule is bound to a substrate. In some embodiments the substrate is selected from: a chip, bead, microfluidic platform, or membrane.

[0011] In some aspects, the present disclosure provides a method of characterizing cardiotoxicity in a subject comprising contacting a biological sample of the subject with a panel comprising capture molecules that bind specifically one or more polypeptide biomarkers selected from the group consisting of: carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosineprotein phosphatase non-receptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain- containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (C0X6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7, and detecting binding of the capture molecule to a polypeptide biomarker present in the biological sample. In some embodiments the method of characterizing cardiotoxicity in a subject comprises contacting a panel comprising capture molecules which bind specifically to carboxypeptidase vitellogenic like (CPVL) and / or polymeric immunoglobulin receptor (PIGR) and detecting binding of the capture molecules to the polypeptide biomarkers present in the biological sample. In some embodiments the method of characterizing cardiotoxicity in a subject comprises contacting a panel comprising capture molecules which bind specifically to carboxypeptidase vitellogenic like (CPVL) and detecting binding of the capture molecules to the polypeptide biomarkers present in the biological sample. In some embodiments, the method comprises detecting an increase in binding of the capture molecule to the polypeptide biomarker relative to a reference, thereby detecting cardiotoxicity in the subject. In some embodiments the capture molecule is an antibody. In some embodiments the capture molecule is an aptamer. In some embodiments the capture molecule of the panel is bound to a substrate. In some embodiments the substrate is selected from: a chip, bead, microfluidic platform, or membrane. In some embodiments the subject has a neoplasia, e.g., a hematological malignancy.

[0012] In another aspect, the present disclosure provides a method of treating a selected subject having a neoplasia, wherein the method comprises administering a cancer therapy agent and a cardioprotective agent to the subject, where the subject is selected by characterizing a biological sample of the subject as having an alteration in the level of a polypeptide biomarker relative to a reference, where the polypeptide biomarker is selected from selected from the group consisting of: carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC- transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase nonreceptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain- containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (C0X6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7, and where the cancer therapy includes an anthracy cline. In some embodiments, the method comprises treating a selected subject having a neoplasia, wherein the method comprises administering a cancer therapy agent and a cardioprotective agent to the subject, where the subject is selected by characterizing a biological sample of the subject as having an increase in the level of carboxypeptidase vitellogenic like (CPVL) and / or polymeric immunoglobulin receptor (PIGR) relative to a reference relative to a reference, where the cancer therapy includes an anthracycline. In some embodiments, the subject is selected where the biological sample has an increase in the level of the polypeptide biomarker relative to a reference. In some embodiments, the cardioprotective agent is an iron chelator, e.g., a bisdioxopiperazine, e.g., dexrazoxane, or a statin, an aldosterone receptor antagonist (MRA), an angiotensin-converting enzyme inhibitor (ACEI), an angiotensin receptor blocker (ARB), and / or a beta-blocker.

[0013] In another aspect, the present disclosure provides a method of treating a selected subject having a neoplasia, wherein the method comprises administering a reduced dosage of a treatment comprising anthracyclines to the subject, where the subject is selected by characterizing a biological sample of the subject as having an alteration in the level of a polypeptide biomarker relative to a reference, where the polypeptide biomarker is selected from the group consisting of: carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC- transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase nonreceptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain- containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7. In some embodiments the method comprises treating a selected subject having a neoplasia, wherein the method comprises administering a reduced dosage of a treatment comprising anthracyclines to the subject, where the subject is selected by characterizing a biological sample of the subject as having an increase in the level of carboxypeptidase vitellogenic like (CPVL) and / or polymeric immunoglobulin receptor (PIGR) relative to a reference. In some embodiments, the subject is selected where the biological sample has an increase in the level of the polypeptide biomarker relative to a reference.

[0014] In another aspect, the present disclosure provides a method of treating a selected subject having a neoplasia, wherein the method comprises administering a cancer therapy agent, where the subject is selected by characterizing a biological sample of the subject as having an alteration in the level of a polypeptide biomarker relative to a reference, where the polypeptide biomarker is selected from selected from the group consisting of: carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase non-receptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain-containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7, and where the cancer therapy agent does not include an anthracycline. In some embodiments, the method comprises treating a selected subject having a neoplasia, wherein the method comprises administering a cancer therapy agent to the subject, where the subject is selected by characterizing a biological sample of the subject as having an increase in the level of carboxypeptidase vitellogenic like (CPVL) and / or polymeric immunoglobulin receptor (PIGR) relative to a reference relative to a reference, where the cancer therapy agent does not include an anthracycline. In some embodiments, the subject is selected where the biological sample has an increase in the level of the polypeptide biomarker relative to a reference.

[0015] In another aspect, the disclosure provides a method of treating a selected subject at risk for heart failure, comprising administering a heart failure treatment, wherein the subject is selected by characterizing a biological sample of the subject as having an alteration in the level of a polypeptide or polynucleotide polypeptide biomarker relative to a reference, wherein the polypeptide biomarker is selected from the group consisting of carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase non-receptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain-containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7. In some embodiments, the selected subject is administered a heart failure treatment selected from a beta-blocker, angiotensin-converting enzyme (ACE) inhibitor, angiotensin receptor blocker (ARB), Sodium-Glucose Cotransporter 2 (SGLT2) inhibitor, and a mineralicorticoid receptor antagonist

[0016] In another aspect, the present disclosure provides a method of detecting cardiotoxicity in a subject, the method comprising detecting a polynucleotide encoding a polypeptide biomarker selected from selected from the group consisting of: carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase non-receptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain- containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7. In another aspect, the present disclosure provides a method of treating a subject having a neoplasia wherein the method comprises administering a treatment including an anthracycline and a cardioprotective agent to the subject, where cardioprotective agent is selected from: SERPINA12; CPVL; PIGR; IQCF3; SESQ2; CSPG2; SHC2; or CFC1 polypeptides or a polynucleotide encoding the polypeptide. In another aspect, the present disclosure provides a method of treating cardiotoxicity associated with anthracycline treatment in a subject having a neoplasia, wherein the method comprises administering a polypeptide, where the polypeptide is: SERPINA12; CPVL; PIGR; IQCF3; SESQ2; CSPG2; SHC2; or CFC1.

[0017] In any of the above aspects, or embodiments thereof, the subject may be treated with an anthracycline or a pharmaceutically acceptable salt thereof.

[0018] In any of the above aspects, or embodiments thereof, a cardioprotective agent may be an administered, e.g., an iron chelator, e.g., a bisdi oxopiperazine, e.g., dexrazoxane. The cardioprotective agent may in some embodiments be selected from a statin, an aldosterone receptor antagonist (MRA), an angiotensin-converting enzyme inhibitor (ACEI), an angiotensin receptor blocker (ARB), and a beta-blocker. In any of the above embodiments, an anthracycline and a cardioprotective agent may be formulated together or separately.

[0019] In any of the above aspects, or embodiments thereof, a biological sample may be a blood sample. In any of the above aspects, or embodiments thereof, a biological sample may be a plasma sample. In any of the above aspects, or embodiments thereof, the subject has a neoplasia.

[0020] In any of the above aspects, or embodiments thereof, the neoplasia may be a cancer, e.g., a hematologic malignancy. In any of the above aspects, or embodiments thereof, the hematologic malignancy may be a leukemia, multiple myeloma (MM), non-Hodgkin lymphoma (NHL), or Hodgkin lymphoma (HL). In any of the above aspects, or embodiments thereof, the hematologic malignancy may be a Diffuse Large B-Cell Lymphoma, Hodgkin’s Lymphoma, Follicular Lymphoma, Acute Myeloid Lymphoma, Non-Hodgkin’s Lymphoma, T-Cell, Histiocyte Rich Large B Cell Lymphoma, or Aggressive B-Cell Lymphoma. In any of the above aspects, or embodiments thereof, the neoplasia may be a sarcoma, breast cancer, stomach cancer, uterine cancer, ovarian cancer, bladder cancer, and / or lung cancer.

[0021] Compositions and articles defined by the disclosure were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the disclosure will be apparent from the detailed description, and from the claims. Definitions

[0022] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0023] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0024] By “agent” is meant a polypeptide, nucleic acid molecule, or small compound.

[0025] By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease or condition.

[0026] By "alteration" is meant a change (an increase or a decrease). As used herein, an alteration includes a 10% change in expression levels, a 25% change, a 40% change, or a 50% or greater change in expression levels.

[0027] By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

[0028] By “anthracy cline” is meant a drug used in cancer therapy that is extracted from Streptomyces bacterium, or a pharmaceutically acceptable salt thereof. Anthracyclines include, without limitation, doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin, ditrisarubicin, and mitoxantrone, and pharmaceutically acceptable salts thereof.

[0029] By "biologic sample" is meant any tissue, cell, fluid, or other material derived from an organism.

[0030] A “biomarker,” e.g., a “polypeptide or polynucleotide biomarker” is differentially present in a biological sample relative to a reference if the mean or median level of the biomarker present in the sample is statistically different from the level present in a reference. A reference level may be, for example, the level present in a sample obtained from a healthy control subject or the level obtained from the subject at an earlier timepoint, i.e., prior to treatment. Common tests for statistical significance include, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann- Whitney and odds ratio. Biomarkers, alone or in combination, provide measures of relative likelihood that a subject belongs to a phenotypic status of interest. Exemplary biomarkers include CPVL, PIGR, biomarkers listed in Table 1, or any other biomarker described herein.

[0031] By "capture reagent" is meant a reagent that binds specifically a nucleic acid molecule or a polypeptide to select or isolate the nucleic acid molecule or polypeptide. In one embodiment, the capture reagent is an aptamer or antibody that binds specifically a polypeptide of interest or a nucleic acid molecule that hybridizes to a polynucleotide encoding a polypeptide of interest.

[0032] In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0033] By “decreases” is meant a reduction by at least about 5% relative to a reference level. A decrease may be by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more and any intervening percentages.

[0034] As used herein, the terms “determining”, “assessing”, “assaying”, “measuring” and “detecting” refer to both quantitative and qualitative determinations, and as such, the term “determining” is used interchangeably herein with “assaying,” “measuring,” and the like. Where a quantitative determination is intended, the phrase “determining an amount” of an analyte and the like is used. Where a qualitative and / or quantitative determination is intended, the phrase “determining a level” of an analyte or “detecting” an analyte is used.

[0035] “Detect” refers to identifying the presence, absence or amount of the analyte to be detected. In embodiments, the analyte is a biomarker described herein.

[0036] By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

[0037] By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In some embodiments, the disease is a neoplasia. In some embodiments, the disease is a cancer, e.g., a sarcoma, a hematological malignancy, breast cancer, stomach cancer, uterine cancer, ovarian cancer, bladder cancer, and / or lung cancer. In some embodiments, the disease is a hematologic malignancy (e.g., leukemia, multiple myeloma (MM), non-Hodgkin lymphoma (NHL), or Hodgkin lymphoma (HL)). In embodiments, the condition is cardiotoxicity.

[0038] By "effective amount" is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. In embodiments, an effective amount of an agent described herein reduces cardiotoxicity (e.g., by at least 5, 10, 25, 50, 75%) or increases cardiac function relative to a reference.

[0039] The term “expression” or “expressed” as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88). Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

[0040] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0041] By “hematologic malignancy” is meant any of a group of diseases involving disruption of normal hematopoietic function and associated with myeloid and lymphatic tumors. Subtypes of hematologic malignancies include but are not limited to leukemia, multiple myeloma (MM), non-Hodgkin lymphoma (NHL), and Hodgkin lymphoma (HL).

[0042] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0043] By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally- occurring genome of the organism from which the nucleic acid molecule of the disclosure is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0044] By an "isolated polypeptide" is meant a polypeptide of the disclosure that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. In an embodiment, the preparation is at least 75%, at least 90%, or at least 99%, by weight, a polypeptide of the disclosure. An isolated polypeptide of the disclosure may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. By “portion” is meant a fragment of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides.

[0045] As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0046] By “reference” is meant a standard or control condition. In some embodiments, the level of a biomarker in a biological sample obtained from a subject having a hematological malignancy is compared to the reference level of the biomarker in a biological sample obtained from a healthy subject. In some embodiments, the reference is the level of biomarker present in a biological sample obtained from a subject having neoplasia, but who does not have and / or who is not at risk for cardiotoxicity after anthracycline treatment. In some embodiments, the reference is obtained from a subject prior to anthracycline administration who does not develop cardiotoxicity after anthracycline treatment. In some embodiments, the reference is obtained from the same subject prior to anthracycline administration.

[0047] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, or even about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

[0048] By "specifically bind" is meant a compound, nucleic acid molecule, or antibody that recognizes and binds a polypeptide of the disclosure, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample.

[0049] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In an embodiment, such a sequence is at least 60%, 80% or 85%, 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

[0050] By "subject" is meant a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, or feline.

[0051] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. As used herein, the terms “treat,” treating,” “treatment,” and the like refer to preventing, reducing, or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0052] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0053] By “vector” is meant a nucleic acid molecule, for example, a plasmid, cosmid, virus, or bacteriophage that is capable of replication in a host cell. In one embodiment, a vector is an expression vector that is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a nucleic acid molecule in a host cell. Typically, expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers.

[0054] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0055] Biomarkers of cardiotoxicity

[0056] In some aspects, provided herein are methods and compositions for characterizing the risk of cardiotoxicity in a biological sample of a subject in need thereof, e.g., prior to administration of an anthracycline. In some embodiments, provided herein are panels comprising capture molecules that bind specifically to an adaptive immune response protein, wherein the alteration of the level of the adaptive immune response protein is associated with a risk of cardiotoxicity in the subject. In some embodiments, the disclosed panels comprise capture molecules that bind specifically to one or more of the polypeptide biomarkers selected from Table 1 or to a polynucleotide encoding such polypeptide biomarkers, below.

[0057] Table 1: Polypeptide biomarkers of cardiotoxicity

[0058] Any of the above protein sequences may be assessed by methods known in the art.

[0059] For example, by “carboxypeptidase vitellogenic like polypeptide” or “CPVL polypeptide” is meant a polypeptide or fragment thereof having at least about 85% or greater sequence identity to the amino acid sequences provided at NCBI Accession Nos.

[0060] NP 001358197.1, NP 001358187.1, NP 001358185.1, NP 001358195.1, NP 001358194.1,

[0061] NP 001358190.1, NP 001358193.1, NP 001358189.1, NP 001358184.1, NP 001358186.1,

[0062] NP 001358191.1, NP 001358192.1, NP 001358196.1, NP 001334983.1, NP 001334981.1,

[0063] NP_061902.2, or NP_112601.3 and having carboxypeptidase activity. Exemplary human CPVL amino acid sequences are provided as SEQ ID NOS: 1-17.

[0064] By “carboxypeptidase vitellogenic like polynucleotide” or “CPVL polynucleotide” is meant a polynucleotide encoding a CPVL polypeptide. Exemplary sequences of CPVL polynucleotides are provided as SEQ ID NOS: 18-34.

[0065] By “cryptic protein polypeptide” or “CFC1 polypeptide” is meant a polypeptide or fragment thereof having at least about 85% or greater sequence identity to the amino acid sequences provided at NCBI Accession Nos. NP_001257349.1, NP_001257350.1, or NP_115934.1 and having activity in signaling during embryonic development. Exemplary human CFC1 amino acid sequences are provided as SEQ ID NOS: 35-37.

[0066] By “cryptic protein polynucleotide” or “CFC1 polynucleotide” is meant a polynucleotide encoding a CFC1 polypeptide. Exemplary sequences of CFC1 polynucleotides are provided as SEQ ID NOS: 38-40.

[0067] By “IQ motif containing F3 polypeptide” or “IQCF3 polypeptide” is meant a polypeptide or fragment thereof having at least about 85% or greater sequence identity to the amino acid sequences provided at NCBI Accession Nos. NP_001078948.1, NP_001193952.1, or NP 001380816.1 and having activity in enabling calmodulin binding. Exemplary human IQCF3 amino acid sequences are provided as SEQ ID NOS: 41-43.

[0068] By “IQ motif containing F3 polynucleotide” or “IQCF3 polynucleotide” is meant a polynucleotide encoding a IQCF3 polypeptide. Exemplary sequences of IQCF3 polynucleotides are provided as SEQ ID NOS: 44-46.

[0069] By “polymeric immunoglobulin receptor polypeptide” or “PIGR polypolypeptide” is meant a polypeptide or fragment thereof having at least about 85% or greater sequence identity to the amino acid sequence provided at NCBI Accession No. NP_002635.2 and having polymeric immunoglobulin molecule binding activity. An exemplary human PIGR amino acid sequence is provided as SEQ ID NO: 47.

[0070] By “polymeric immunoglobulin receptor polynucleotide” or “PIGR polynucleotide” is meant a polynucleotide encoding a PIGR polypeptide. An exemplary sequence of a PIGR polynucleotide is provided as SEQ ID NO: 48.

[0071] By “serpin family A member 12 polypeptide” or “SERPINA12 polypeptide” is meant a polypeptide or fragment thereof having at least about 85% or greater sequence identity to the amino acid sequence provided at NCBI Accession No. NP_001291390.1 or NP_776249.1 and having negative regulation of endopeptidase activity. Exemplary human SERPINA12 amino acid sequences are provided as SEQ ID NOS: 49-50.

[0072] By “serpin family A member 12 polynucleotide” or “SERPINA12 polynucleotide” is meant a polynucleotide encoding a SERPINA12 polypeptide. Exemplary sequences of SERPINA12 polynucleotides are provided as SEQ ID NOS: 51-52.

[0073] By “Sesquipedalian-2 polypeptide” or “SESQ2 polypeptide,” also known as PH domain containing endocytic trafficking adaptor 2 (PHETA2), is meant a polypeptide or fragment thereof having at least about 85% or greater sequence identity to the amino acid sequence provided at NCBI Accession No. NP_001002034.2 and having endosome organization activity. An exemplary human SESQ2 amino acid sequence is provided as SEQ ID NO: 53.

[0074] By “Sesquipedalian-2 polypeptide” or “SESQ2 polypeptide,” also known as PH domain containing endocytic trafficking adaptor 2 (PHETA2) polynucleotide, is meant a polynucleotide encoding a SESQ2 polypeptide. An exemplary sequence of a SESQ2 polynucleotide is provided as SEQ ID NO: 54.

[0075] By “SHC -transforming protein 2 polypeptide” or “SHC2 polypeptide” is meant a polypeptide or fragment thereof having at least about 85% or greater sequence identity to the amino acid sequences provided at NCBI Accession No. NP_001373985.1 or NP_036567.2 and having activity in enabling receptor tyrosine kinase binding. Exemplary human SHC2 amino acid sequences are provided as SEQ ID NOS: 55-56.

[0076] By “SHC-transforming protein 2 polynucleotide” or “SHC2 polynucleotide” is meant a polynucleotide encoding a SHC2 polypeptide. Exemplary sequences of SHC2 polynucleotides are provided as SEQ ID NOS: 57-58.

[0077] By “versican core protein polypeptide” or “CSPG2 polypeptide,” also known as VCAN, is meant a polypeptide or fragment thereof having at least about 85% or greater sequence identity to the amino acid sequences provided at NCBI Accession Nos. NP_001119808.1, NP_001157569.1, NP_001157570.1, or NP_004376.2 and having cell adhesion, proliferation, migration, and / or angiogenesis activity. Exemplary human CSPG2 amino acid sequences are provided as SEQ ID NOS: 59-62.

[0078] By “versican core protein polynucleotide” or “CSPG2 polynucleotide” is meant a polynucleotide encoding a CSPG2 polypeptide. Exemplary sequences of CSPG2 polynucleotides are provided as SEQ ID NOS: 63-66. Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0079] BRIEF DESCRIPTION OF THE FIGURES

[0080] FIG. 1 is a schematic of the clinical study timeline described in Examples 1 and 2, in which proteomics analysis was performed prior to the administration of anthracycline. The levels of the top polypeptide biomarker hits correlated with cardiac function post-anthracycline administration.

[0081] FIGS. 2A-2F show the relationship between the relative change in left ventricular ejection fraction (AEF) and poly-immunoglobulin receptor (PIGR) or IgA levels in the clinical studies described in Examples 1 and 2. FIG. 2A is a graph showing the relative change in EF relative to circulating PIGR in the discovery cohort proteomics assay. FIG. 2B is a graph showing the relative change in EF relative to circulating PIGR in a discovery cohort ELISA assay. FIG. 2C is a graph showing the relative change in EF relative to circulating PIGR in a validation cohort ELISA assay. FIG. 2D is a graph showing the relative change in EF relative to circulating IgA in the discovery cohort ELISA assay. FIG. 2E is a graph showing the relative change in EF relative to circulating IgA in the validation cohort ELISA assay. FIG. 2F is a graph showing the relationship between circulating IgA and circulating PIGR in the discovery and validation cohorts.

[0082] FIGS. 3A-3H show the effects of doxorubicin in mice on cardiac function, plasma PIGR, and circulating IgA with doxorubicin (Dox) administration, as described in Example 2. FIG. 3A is a schematic of the mouse study in which Dox was administered over the course of the 8 week study. FIG. 3B is a graph showing the fractional shortening that occurs with Dox at the 4 week and 8 week time points of the mouse study. FIG. 3C is a graph showing the change in internal diameter of the left ventricle that occurs with Dox at the 4 week and 8 week time points of the mouse study. FIG. 3D is a graph showing the change in mouse plasma PIGR levels with Dox administration. FIG. 3E is a graph showing mouse fractional shortening relative to circulating PIGR with Dox administration. FIG. 3F is a graph showing the relationship between mouse fractional shortening and change in internal diameter of the left ventricle with Dox administration. FIG. 3G is a graph showing the levels of circulating CD19+B-cells with Dox at the 4 week and 8 week time points of the mouse study. FIG. 3H is a graph showing the levels of circulating IgA+CD19+B-cells with Dox at the 4 week and 8 week time points of the mouse study.

[0083] FIGS. 4A-4J show the relationship between carboxypeptidase vitellogenic-like protein (CPVL) and cardiac function in patients and mice. FIG. 4A is a graph showing the relative change in left ventricular ejection fraction (AEF) and CPVL levels in the clinical discovery cohort proteomics assay. FIG. 4B is an immunoblot showing the CPVL levels in discovery cohort patients with no change in EF and with a greater than 20% decline in AEF. FIG. 4C is an immunoblot showing the CPVL levels in validation cohort patients with no change in EF and with a greater than 20% decline in AEF. FIG. 4D is a graph showing the quantification of the assay depicted in FIG. 4B assays. FIG. 4E is a graph showing the quantification of the assay depicted in FIG 4C. FIG. 4F is an immunoblot showing plasma CPVL levels in mice treated with Dox at the 8 week timepoint. FIG. 4G is an immunoblot showing whole cardiac lysate CPVL levels in mice treated with Dox at the 8 week timepoint. FIG. 4H is a graph showing quantification of the assay depicted in FIG. 4F. FIG. 41 is a graph showing quantification of the assay depicted in FIG. 4G. FIG. 4J is a graph showing the relationship between plasma CPVL levels and fractional shortening in the mice, where the data points from the Dox treated mice are circled.

[0084] FIGS. 5A-5B show the relationship in the discovery cohort between change in LVEF and circulating PIGR or CPVL when the patient that developed heart failure is excluded from the results. FIG. 5A is a graph showing the relative change in EF versus the PIGR levels of the discovery cohort with the patient exclusion. FIG. 5B is a graph showing the relative change in EF versus the CPVL levels of the discovery cohort with the patient exclusion.

[0085] FIGS. 6A-6C show the effects on Dox on cardiac B-cells as measured by flow cytometry. FIG. 6A is a set of graphs depicting the gating strategy of the flow cytometry experiments performed with blood (top) and heart (bottom) B-cells. FIG. 6B is a graph of the cardiac CD19+B-cells in mice treated with Dox at the 4 week and 8 week timepoints. FIG. 6C is a graph of the cardiac IgA+CD19+B-cells in mice treated with Dox at the 4 week and 8 week timepoints.

[0086] FIG. 7 is an immunoblot of CPVL in bone marrow derived macrophages and CD8+ T- cell lysates. FIG. 8 provides immunoblots of CPVL in the lysate (left) and supernatant (right) of Thpl derived macrophages treated with increasing doses of Dox.

[0087] DETAILED DESCRIPTION OF THE INVENTION

[0088] Provided herein are compositions and methods for detecting, treating, e.g., preventing or reducing, cardiotoxicity in a subject (e.g., a subject at risk of anthracycline cardiotoxicity). Anthracy cline cancer therapy serves as the backbone for many first-line regimens for hematologic malignancy and other cancers. However, anthracyclines cause cardiotoxicity in a significant minority of patients. The present disclosure describes inter alia the discovery of plasma sample biomarkers from cancer patients predictive of cardiotoxicity. The biomarkers provided herein, and as shown in Tables 1 and 6, correlated with a later decrease in cardiac function after the patient was administered anthracycline, and thus may be used to determine the risk of a subject in developing a cardiotoxicity.

[0089] In some aspects, the disclosure provides panels for characterizing the risk of cardiotoxicity in a biological sample of a subject in need of anthracycline treatment, e.g., for the treatment of a neoplasia, prior to administration of an anthracycline. In some aspects, the panels comprise capture molecules that bind specifically to adaptive immune response proteins, wherein the risk of developing cardiotoxicity is associated with an alteration, e.g, an increase, in the level of the adaptive immune response protein.

[0090] In some embodiments, the panels disclosed herein comprise capture molecules that bind specifically to a biomarker as provided in Table 1, e.g., carboxypeptidase vitellogenic like (CPVL). In some embodiments, the panels disclosed herein comprise capture molecules that bind specifically to a biomarker selected from: carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase non-receptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF- 16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain- containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7.

[0091] In some embodiments, a subject is selected for a treatment when the biological sample of the subject has an increased binding in the capture molecule and the polypeptide biomarker (e.g., of Table 1) relative to a reference, thereby detecting cardiotoxicity or a propensity to develop cardiotoxicity in the subject.

[0092] In some embodiments, a subject in need of treating, e.g., preventing or reducing, cardiotoxicity with anthracy cline treatment has a cancer, e.g., a hematological malignancy selected from leukemia, multiple myeloma (MM), Diffuse Large B-Cell Lymphoma, Hodgkin’s Lymphoma, Follicular Lymphoma, Acute Myeloid Lymphoma, Non-Hodgkin’s Lymphoma, T- Cell, Histiocyte Rich Large B Cell Lymphoma, or Aggressive B-Cell Lymphoma. In some embodiments, a subject in need of treating, e.g., preventing or reducing, cardiotoxicity with anthracycline treatment has a sarcoma, breast cancer, stomach cancer, uterine cancer, ovarian cancer, bladder cancer, and / or lung cancer.

[0093] Anthracyclines

[0094] Anthracyclines are a class of drugs used in cancer therapy, extracted from Streptomyces bacterium, that are effective first line therapies for a number of cancers, including leukemias, lymphomas, sarcomas, breast, stomach, uterine, ovarian, bladder cancer, and lung cancers. However, in a significant minority of patients, anthracyclines cause cardiotoxicity. The compositions and methods provided herein may be used to treat, e.g., prevent or reduce, cardiotoxicity related to anthracycline administration. In some embodiments, a selected subject is administered an anthracycline selected from doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin, ditrisarubicin, and mitoxantrone, or a pharmaceutically acceptable salt thereof. In some embodiments a selected subject is administered a reduced dose of anthracycline, an anthracycline treatment in combination with a cancer therapy that is not cytotoxic, or an anthracycline treatment in combination with a cardioprotective agent. Selected subjects are those identified as at risk of having or having a propensity to develop anthracycline cardiotoxicity. Such subjects are selected by characterizing the level of a biomarker (e.g., PIGR, CPVL, or a biomarker of Table 1) in a biological sample of the subject relative to a reference as described herein.

[0095] Doxorubicin Doxorubicin interacts with DNA by intercalation and inhibition of macromolecular biosynthesis. This inhibits the progression of topoisomerase II, an enzyme which relaxes supercoils in DNA for transcription. Doxorubicin stabilizes the topoisomerase II complex after it has broken the DNA chain for replication, preventing the DNA double helix from being released and thereby stopping the process of replication. It may also increase quinone type free radical production, hence contributing to its cytotoxicity.

[0096] Daunorubicin

[0097] Similar to doxorubicin, daunorubicin interacts with DNA by intercalation and inhibition of macromolecular biosynthesis. This inhibits the progression of the enzyme topoisomerase II, which relaxes supercoils in DNA; without action of topoisomerase II, these DNA supercoils interfere in transcription of DNA. Daunorubicin stabilizes the topoisomerase II complex after it has broken the DNA chain for replication, preventing the DNA double helix from being resealed and thereby stopping the process of replication. It can also induce histone eviction from chromatin upon intercalation contributing to its cytotoxicity.

[0098] Epirubicin

[0099] The mechanism of action of epirubicin is similar to that of doxorubicin and other anthracycline drugs. The observed clinical differences between epirubicin and doxorubicin can be explained by the pharmacokinetic differences based on the different affinity to DNA and lipophilicity, as there is no indication that different mechanisms are involved in their activity.

[0100] Epirubicin exhibits activity in all phases of the cell cycle, but maximal cell kill occurs during the S phase and G2 phase of the cell cycle.

[0101] Idarubicin

[0102] Idarubicin inserts itself into DNA and prevents DNA unwinding by interfering with the enzyme topoisomerase II. It is an analog of daunorubicin, but the absence of a methoxy group increases its fat solubility and cellular uptake. Similar to other anthracyclines, it also induces histone eviction from chromatin.

[0103] Mitoxantrone

[0104] Mitoxantrone is a type II topoisomerase inhibitor; it disrupts DNA synthesis and DNA repair in both healthy cells and cancer cells by intercalation between DNA bases.

[0105] Cardiotoxicity of Anthracyclines

[0106] Cardiotoxicity can occur during or after the completion of treatment with anthracyclines, and may become apparent within one year of the completion of treatment (early onset chronic cardiotoxicity) or many years after cancer therapy has been completed (late onset chronic cardiotoxicity). Up to 65% of patients with a history of a childhood malignancy treated with doxorubicin may have echocardiographic evidence of left ventricular contractile abnormalities. In a previous study involving analysis of 14,358 5-year survivors of childhood malignancies, use of less than 250 mg / m2of anthracycline was associated with a 2.4-fold higher risk of developing congestive heart failure compared to those patients who did not receive anthracyclines (Mulrooney et al., 2009, Bmj 339:b4606). This risk increased to 5.2-fold with the use of greater than or equal to 250 mg / m2of doxorubicin.

[0107] In addition to this late cardiotoxicity, a rare form of acute anthracycline cardiotoxicity has been previously described (Bristow et al., 1978, Am J Med. 65(5): 823-32). The manifestations of this potentially lethal cardiotoxicity may include pericarditis and arrhythmias in addition to left ventricular dysfunction.

[0108] Biomarker Isolation

[0109] In some embodiments, the disclosure provides panels comprising isolated polypeptide biomarkers (carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase non-receptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain- containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7) or capture markers binding such polypeptide biomarkers. In some embodiments, the disclosure provides panels comprising polynucleotides (e.g., probes, primers) that bind a polynucleotide encoding a polypeptide biomarker described herein.

[0110] The biomarkers may be isolated from biological fluids, such as urine, plasma, or serum. They can be isolated by any method known in the art. In certain embodiments, this isolation is accomplished using the mass and / or binding characteristics of the markers. For example, a sample comprising the biomolecules can be subject to chromatographic fractionation and subject to further separation by, e.g., acrylamide gel electrophoresis. Knowledge of the identity of the biomarker also allows their isolation by immunoaffinity chromatography. By “isolated polypeptide biomarker” is meant at least 60%, by weight, free from proteins and naturally- occurring organic molecules with which the marker is naturally associated. In some embodiments, the preparation is at least 75, 80, 85, 90 or 95% pure or at least 99%, by weight, a purified marker.

[0111] The biomarkers of this disclosure (e.g., carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase non-receptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain- containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7) can be detected by any suitable method. The methods described herein can be used individually or in combination for a more accurate detection of the biomarkers (e.g., biochip in combination with mass spectrometry, immunoassay in combination with mass spectrometry, and the like).

[0112] Detection paradigms that can be employed in the disclosure include, but are not limited to, optical methods, electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy. Illustrative of optical methods, in addition to microscopy, both confocal and non-confocal, are detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry). These and additional methods are described infra.

[0113] Detection by Immunoassay

[0114] In particular embodiments, the polypeptide biomarkers of the disclosure (e.g., carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC- transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase nonreceptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain- containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7) are measured by immunoassay. Immunoassay typically utilizes an antibody or fragment thereof detect the presence or level of a polypeptide biomarker in a sample. Antibodies can be produced by methods well known in the art, e.g., by immunizing animals with the polypeptide biomarkers. Polypeptide biomarkers can be isolated from samples based on their binding characteristics. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies by methods well known in the art.

[0115] This disclosure contemplates traditional immunoassays including, for example, Western blot, sandwich immunoassays including ELISA and other enzyme immunoassays, fluorescencebased immunoassays, and chemiluminescence. Nephelometry is an assay done in liquid phase, in which antibodies are in solution. Binding of the antigen to the antibody results in changes in absorbance, which is measured. Other forms of immunoassay include magnetic immunoassay, radioimmunoassay, and real-time immunoquantitative PCR (iqPCR).

[0116] Immunoassays can be carried out on solid substrates (e.g., chips, beads, microfluidic platforms, membranes) or on any other forms that supports binding of the antibody to the marker and subsequent detection. A single marker may be detected at a time or a multiplex format may be used. Multiplex immunoanalysis may involve planar microarrays (protein chips) and bead-based microarrays (suspension arrays).

[0117] In a SELDLbased immunoassay, a biospecific capture reagent for the polypeptide biomarker is attached to the surface of an MS probe, such as a pre-activated ProteinChip array. The polypeptide biomarker is then specifically captured on the biochip through this reagent, and the captured polypeptide biomarker is detected by mass spectrometry.

[0118] Detection by Biochip

[0119] In aspects of the disclosure, a sample is analyzed by means of a biochip (also known as a microarray). The polypeptides and nucleic acid molecules of the disclosure are useful as hybridizable array elements in a biochip. Biochips generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there.

[0120] The array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate. Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins. Methods for making nucleic acid microarrays are known to the skilled artisan and are described, for example, in U.S. Pat. No. 5,837,832, Lockhart, et al. (Nat. Biotech. 14:1675-1680, 1996), and Schena, et al. (Proc. Natl. Acad. Sci. 93: 10614-10619, 1996), herein incorporated by reference. Methods for making polypeptide microarrays are described, for example, by Ge (Nucleic Acids Res. 28: e3. i-e3. vii, 2000), MacBeath et al., (Science 289:1760-1763, 2000), Zhu et al. (Nature Genet. 26:283-289), and in U.S. Pat. No. 6,436,665, hereby incorporated by reference.

[0121] Detection by Protein Biochip

[0122] In aspects of the disclosure, a sample is analyzed by means of a protein biochip (also known as a protein microarray). Such biochips are useful in high-throughput low-cost screens to identify alterations in the expression or post-translation modification of a polypeptide of the disclosure, or a fragment thereof. In embodiments, a protein biochip of the disclosure binds a polypeptide biomarker present in a subject sample and detects an alteration in the level of the polypeptide biomarker. Typically, a protein biochip features a protein, or fragment thereof, bound to a solid support. Suitable solid supports include membranes (e.g., membranes composed of nitrocellulose, paper, or other material), polymer-based films (e.g., polystyrene), beads, or glass slides. For some applications, proteins (e.g., antibodies that bind a marker of the disclosure) are spotted on a substrate using any convenient method known to the skilled artisan (e.g., by hand or by inkjet printer).

[0123] In embodiments, the protein biochip is hybridized with a detectable probe. Such probes can be polypeptide, nucleic acid molecules, antibodies, or small molecules. For some applications, polypeptide and nucleic acid molecule probes are derived from a biological sample taken from a patient, such as a bodily fluid (such as blood, blood serum, plasma, saliva, urine, ascites, cyst fluid, and the like); a homogenized tissue sample (e.g., a tissue sample obtained by biopsy); or a cell isolated from a patient sample. Probes can also include antibodies, candidate peptides, nucleic acids, or small molecule compounds derived from a peptide, nucleic acid, or chemical library. Hybridization conditions (e.g., temperature, pH, protein concentration, and ionic strength) are optimized to promote specific interactions. Such conditions are known to the skilled artisan and are described, for example, in Harlow, E. and Lane, D., Using Antibodies : A Laboratory Manual. 1998, New York: Cold Spring Harbor Laboratories. After removal of nonspecific probes, specifically bound probes are detected, for example, by fluorescence, enzyme activity (e.g., an enzyme-linked calorimetric assay), direct immunoassay, radiometric assay, or any other suitable detectable method known to the skilled artisan.

[0124] Many protein biochips are described in the art. These include, for example, protein biochips produced by Ciphergen Biosystems, Inc. (Premont, CA), Zyomyx (Hayward, CA), Packard BioScience Company (Meriden, CT), Phylos (Lexington, MA), Invitrogen (Carlsbad, CA), Biacore (Uppsala, Sweden) and Procognia (Berkshire, UK). Examples of such protein biochips are described in the following patents or published patent applications: U.S. Patent Nos. 6,225,047; 6,537,749; 6,329,209; and 5,242,828; PCT International Publication Nos. WO 00 / 56934; WO 03 / 048768; and WO 99 / 51773.

[0125] Detection by Nucleic Acid Biochip

[0126] In aspects of the disclosure, a sample is analyzed by means of a nucleic acid biochip (also known as a nucleic acid microarray). To produce a nucleic acid biochip, oligonucleotides may be synthesized or bound to the surface of a substrate using a chemical coupling procedure and an inkjet application apparatus, as described in PCT application W095 / 251116 (Baldeschweiler et al.). Alternatively, a gridded array may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedure.

[0127] A nucleic acid molecule (e.g. RNA or DNA) derived from a biological sample may be used to produce a hybridization probe as described herein. The biological samples are generally derived from a patient, e.g., as a bodily fluid (such as blood, blood serum, plasma, saliva, urine, ascites, cyst fluid, and the like); a homogenized tissue sample (e.g., a tissue sample obtained by biopsy); or a cell isolated from a patient sample. Lor some applications, cultured cells or other tissue preparations may be used. The mRNA is isolated according to standard methods, and cDNA is produced and used as a template to make complementary RNA suitable for hybridization. Such methods are well known in the art. The RNA is amplified in the presence of fluorescent nucleotides, and the labeled probes are then incubated with the microarray to allow the probe sequence to hybridize to complementary oligonucleotides bound to the biochip.

[0128] Incubation conditions are adjusted such that hybridization occurs with precise complementary matches or with various degrees of less complementarity depending on the degree of stringency employed. For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, of at least about 37°C., or of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In one embodiment, hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In embodiments, hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In other embodiments, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0129] The removal of nonhybridized probes may be accomplished, for example, by washing. The washing steps that follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25°C, of at least about 42°C, or of at least about 68°C. In embodiments, wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In other embodiments, wash steps will occur at 68 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.

[0130] Detection system for measuring the absence, presence, and amount of hybridization for all of the distinct nucleic acid sequences are well known in the art. For example, simultaneous detection is described in Heller et al., Proc. Natl. Acad. Sci. 94:2150-2155, 1997. In embodiments, a scanner is used to determine the levels and patterns of fluorescence.

[0131] Detection by Mass Spectrometry

[0132] In aspects, the polypeptide biomarkers are detected by mass spectrometry (MS). Mass spectrometry is a well known tool for analyzing chemical compounds that employs a mass spectrometer to detect gas phase ions. Mass spectrometers are well known in the art and include, but are not limited to, time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. The method may be performed in an automated (Villanueva, etal., Nature Protocols (2006) l(2):880-891) or semi-automated format. This can be accomplished, for example with the mass spectrometer operably linked to a liquid chromatography device (LC-MS / MS or LC-MS) or gas chromatography device (GC-MS or GC-MS / MS). Methods for performing mass spectrometry are well known and have been disclosed, for example, in US Patent Application Publication Nos: 20050023454; 20050035286; US Patent No. 5,800,979 and the references disclosed therein.

[0133] Laser Desorption / ionization

[0134] In embodiments, the mass spectrometer is a laser desorption / ionization mass spectrometer. In laser desorption / ionization mass spectrometry, the analytes are placed on the surface of a mass spectrometry probe, a device adapted to engage a probe interface of the mass spectrometer and to present an analyte to ionizing energy for ionization and introduction into a mass spectrometer. A laser desorption mass spectrometer employs laser energy, typically from an ultraviolet laser, but also from an infrared laser, to desorb analytes from a surface, to volatilize and ionize them and make them available to the ion optics of the mass spectrometer. The analysis of proteins by LDI can take the form of MALDI or of SELDI. The analysis of proteins by LDI can take the form of MALDI or of SELDI.

[0135] Laser desorption / ionization in a single time of flight instrument typically is performed in linear extraction mode. Tandem mass spectrometers can employ orthogonal extraction modes.

[0136] Matrix-assisted Laser Desorption / ionization (MALDI) and Electrospray Ionization (ESI)

[0137] In embodiments, the mass spectrometric technique for use in the disclosure is matrix- assisted laser desorption / ionization (MALDI) or electrospray ionization (ESI). In related embodiments, the procedure is MALDI with time of flight (TOF) analysis, known as MALDL TOF MS. This involves forming a matrix on a membrane with an agent that absorbs the incident light strongly at the particular wavelength employed. The sample is excited by UV or IR laser light into the vapor phase in the MALDI mass spectrometer. Ions are generated by the vaporization and form an ion plume. The ions are accelerated in an electric field and separated according to their time of travel along a given distance, giving a mass / charge (m / z) reading which is very accurate and sensitive. MALDI spectrometers are well known in the art and are commercially available from, for example, PerSeptive Biosystems, Inc. (Framingham, Mass., USA).

[0138] Magnetic-based serum processing can be combined with traditional MALDI-TOF. Through this approach, improved peptide capture is achieved prior to matrix mixture and deposition of the sample on MALDI target plates. Accordingly, in embodiments, methods of peptide capture are enhanced through the use of derivatized magnetic bead based sample processing.

[0139] MALDI-TOF MS allows scanning of the fragments of many proteins at once. Thus, many proteins can be run simultaneously on a polyacrylamide gel, subjected to a method of the disclosure to produce an array of spots on a collecting membrane, and the array may be analyzed. Subsequently, automated output of the results is provided by using an server (e.g., ExPASy) to generate the data in a form suitable for computers.

[0140] Other techniques for improving the mass accuracy and sensitivity of the MALDI-TOF MS can be used to analyze the fragments of protein obtained on a collection membrane. These include, but are not limited to, the use of delayed ion extraction, energy reflectors, ion-trap modules, and the like. In addition, post source decay and MS -MS analysis are useful to provide further structural analysis. With ESI, the sample is in the liquid phase and the analysis can be by ion-trap, TOF, single quadrupole, multi-quadrupole mass spectrometers, and the like. The use of such devices (other than a single quadrupole) allows MS-MS or MSnanalysis to be performed. Tandem mass spectrometry allows multiple reactions to be monitored at the same time.

[0141] Capillary infusion may be employed to introduce the marker to a desired mass spectrometer implementation, for instance, because it can efficiently introduce small quantities of a sample into a mass spectrometer without destroying the vacuum. Capillary columns are routinely used to interface the ionization source of a mass spectrometer with other separation techniques including, but not limited to, gas chromatography (GC) and liquid chromatography (LC). GC and LC can serve to separate a solution into its different components prior to mass analysis. Such techniques are readily combined with mass spectrometry. One variation of the technique is the coupling of high performance liquid chromatography (HPLC) to a mass spectrometer for integrated sample separation / and mass spectrometer analysis.

[0142] Quadrupole mass analyzers may also be employed as needed to practice the disclosure. Fourier-transform ion cyclotron resonance (FTMS) can also be used for some disclosure embodiments. It offers high resolution and the ability of tandem mass spectrometry experiments. FTMS is based on the principle of a charged particle orbiting in the presence of a magnetic field. Coupled to ESI and MALDI, FTMS offers high accuracy with errors as low as 0.001%.

[0143] Surface-enhanced laser desorption / ionization (SELDI)

[0144] In embodiments, the mass spectrometric technique for use in the disclosure is “Surface Enhanced Laser Desorption and Ionization” or “SELDI,” as described, for example, in U.S. Patents No. 5,719,060 and No. 6,225,047, both to Hutchens and Yip. This refers to a method of desorption / ionization gas phase ion spectrometry (e.g., mass spectrometry) in which an analyte (here, one or more of the polypeptide biomarkers) is captured on the surface of a SELDI mass spectrometry probe.

[0145] SELDI has also been called “affinity capture mass spectrometry.” It also is called “Surface-Enhanced Affinity Capture” or “SEAC”. This version involves the use of probes that have a material on the probe surface that captures analytes through a non-covalent affinity interaction (adsorption) between the material and the analyte. The material is variously called an “adsorbent,” a “capture reagent,” an “affinity reagent” or a “binding moiety.” Such probes can be referred to as “affinity capture probes” and as having an “adsorbent surface.” The capture reagent can be any material capable of binding an analyte. The capture reagent is attached to the probe surface by physisorption or chemisorption. In certain embodiments the probes have the capture reagent already attached to the surface. In other embodiments, the probes are preactivated and include a reactive moiety that is capable of binding the capture reagent, e.g., through a reaction forming a covalent or coordinate covalent bond. Epoxide and acyl-imidizole are useful reactive moieties to covalently bind polypeptide capture reagents such as antibodies or cellular receptors. Nitrilotriacetic acid and iminodiacetic acid are useful reactive moieties that function as chelating agents to bind metal ions that interact non- covalently with histidine containing peptides. Adsorbents are generally classified as chromatographic adsorbents and biospecific adsorbents.

[0146] “Chromatographic adsorbent” refers to an adsorbent material typically used in chromatography. Chromatographic adsorbents include, for example, ion exchange materials, metal chelators (e.g., nitrilotriacetic acid or iminodiacetic acid), immobilized metal chelates, hydrophobic interaction adsorbents, hydrophilic interaction adsorbents, dyes, simple biomolecules (e.g., nucleotides, amino acids, simple sugars and fatty acids) and mixed mode adsorbents (e.g., hydrophobic attraction / electrostatic repulsion adsorbents).

[0147] “Biospecific adsorbent” refers to an adsorbent comprising a biomolecule, e.g., a nucleic acid molecule (e.g., an aptamer), a polypeptide, a polysaccharide, a lipid, a steroid or a conjugate of these (e.g, a glycoprotein, a lipoprotein, a glycolipid, a nucleic acid (e.g., DNA)-protein conjugate). In certain instances, the biospecific adsorbent can be a macromolecular structure such as a multiprotein complex, a biological membrane or a virus. Examples of biospecific adsorbents are antibodies, receptor proteins and nucleic acids. Biospecific adsorbents typically have higher specificity for a target analyte than chromatographic adsorbents. Further examples of adsorbents for use in SELDI can be found in U.S. Patent No. 6,225,047. A “bioselective adsorbent” refers to an adsorbent that binds to an analyte with an affinity of at least 10'8M.

[0148] Protein biochips produced by Ciphergen comprise surfaces having chromatographic or biospecific adsorbents attached thereto at addressable locations. Ciphergen’s ProteinChip® arrays include NP20 (hydrophilic); H4 and H50 (hydrophobic); SAX-2, Q-10 and (anion exchange); WCX-2 and CM-10 (cation exchange); IMAC-3, IMAC-30 and IMAC-50 (metal chelate);and PS- 10, PS-20 (reactive surface with acyl-imidizole, epoxide) and PG-20 (protein G coupled through acyl-imidizole). Hydrophobic ProteinChip arrays have isopropyl or nonylphenoxy-poly(ethylene glycol)methacrylate functionalities. Anion exchange ProteinChip arrays have quaternary ammonium functionalities. Cation exchange ProteinChip arrays have carboxylate functionalities. Immobilized metal chelate ProteinChip arrays have nitrilotriacetic acid functionalities (IMAC 3 and IMAC 30) or O-methacryloyl-N,N-bis-carboxymethyl tyrosine functionalities (IMAC 50) that adsorb transition metal ions, such as copper, nickel, zinc, and gallium, by chelation. Preactivated ProteinChip arrays have acyl-imidizole or epoxide functional groups that can react with groups on proteins for covalent binding.

[0149] Such biochips are further described in: U.S. Patent No. 6,579,719 (Hutchens and Yip, “Retentate Chromatography,” June 17, 2003); U.S. Patent 6,897,072 (Rich et al., “Probes for a Gas Phase Ion Spectrometer,” May 24, 2005); U.S. Patent No. 6,555,813 (Beecher et al., “Sample Holder with Hydrophobic Coating for Gas Phase Mass Spectrometer,” April 29, 2003); U.S. Patent Publication No. U.S. 2003 -0032043 Al (Pohl and Papanu, “Latex Based Adsorbent Chip,” July 16, 2002); and PCT International Publication No. WO 03 / 040700 (Um et al., “Hydrophobic Surface Chip,” May 15, 2003); U.S. Patent Application Publication No. US 2003 / -0218130 Al (Boschetti et al., “Biochips With Surfaces Coated With Polysaccharide- Based Hydrogels,” April 14, 2003) and U.S. Patent 7,045,366 (Huang et al., “Photocrosslinked Hydrogel Blend Surface Coatings” May 16, 2006).

[0150] In general, a probe with an adsorbent surface is contacted with the sample for a period of time sufficient to allow the polypeptide biomarker that may be present in the sample to bind to the adsorbent. After an incubation period, the substrate is washed to remove unbound material. Any suitable washing solutions can be used. In an embodimentaqueous solutions are employed. The extent to which molecules remain bound can be manipulated by adjusting the stringency of the wash. The elution characteristics of a wash solution can depend, for example, on pH, ionic strength, hydrophobicity, degree of chaotropism, detergent strength, and temperature. Unless the probe has both SEAC and SEND properties (as described herein), an energy absorbing molecule then is applied to the substrate with the bound polypeptide biomarkers.

[0151] In yet another method, one can capture the polypeptide biomarkers with a solid-phase bound immuno-adsorbent that has antibodies that bind the polypeptide biomarkers. After washing the adsorbent to remove unbound material, the polypeptide biomarkers are eluted from the solid phase and detected by applying to a SELDI biochip that binds the polypeptide biomarkers and analyzing by SELDI.

[0152] The polypeptide biomarkers bound to the substrates are detected in a gas phase ion spectrometer such as a time-of-flight mass spectrometer. The polypeptide biomarkers are ionized by an ionization source such as a laser, the generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions. The detector then translates information of the detected ions into mass-to-charge ratios. Detection of a polypeptide biomarker typically will involve detection of signal intensity. Thus, both the quantity and mass of the polypeptide biomarker can be determined.

[0153] Antibodies

[0154] As reported herein, antibodies that bind specifically a biomarker (e.g., carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase non-receptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain-containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7) are useful in the methods of the disclosure, including methods of detection and therapeutic methods. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Tetramers may be naturally occurring or reconstructed from single chain antibodies or antibody fragments. As used herein, the term “antibody” means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogenbinding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , and Fv fragments, linear antibodies, scFv antibodies, single-domain antibodies, such as camelid antibodies (Riechmann, 1999, Journal of Immunological Methods 231:25-38), composed of either a VL or a VH domain which exhibit sufficient affinity for the target, and multispecific antibodies formed from antibody fragments.

[0155] The antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab') 2 , as well as single chain antibodies (scFv), humanized antibodies, and human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). For example, F(ab')2, and Fab fragments that lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983). Thus, the antibodies comprise, without limitation, whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab’, single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies.

[0156] Unconventional antibodies include, but are not limited to, nanobodies, linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062,1995), single domain antibodies, single chain antibodies, and antibodies having multiple valencies ( .g., diabodies, tribodies, tetrabodies, and pentabodies). Nanobodies are the smallest fragments of naturally occurring heavy-chain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies have the affinity and specificity of conventional antibodies although they are only half of the size of a single chain Fv fragment. The consequence of this unique structure, combined with their extreme stability and a high degree of homology with human antibody frameworks, is that nanobodies can bind therapeutic targets not accessible to conventional antibodies. Recombinant antibody fragments with multiple valencies provide high binding avidity and unique targeting specificity to cancer cells. These multimeric scFvs ( .g., diabodies, tetrabodies) offer an improvement over the parent antibody since small molecules of ~60-100kDa in size provide faster blood clearance and rapid tissue uptake See Power et al., (Generation of recombinant multimeric antibody fragments for tumor diagnosis and therapy. Methods Mol Biol, 207, 335-50, 2003); and Wu et al. (Anti-carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging. Tumor Targeting, 4, 47-58, 1999). Various techniques for making and using unconventional antibodies have been described. Bispecific antibodies produced using leucine zippers are described by Kostelny et al. (J. Immunol. 148(5):1547-1553, 1992). Diabody technology is described by Hollinger et al. (Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993). Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) diners is described by Gruber et al. (J. Immunol. 152:5368, 1994). Trispecific antibodies are described by Tutt et al. (J. Immunol. 147:60, 1991). Single chain Fv polypeptide antibodies include a covalently linked VH: : VL heterodimer which can be expressed from a nucleic acid including VH- and Vt-encoding sequences either joined directly or joined by a peptide-encoding linker as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754.

[0157] In various embodiments, an antibody is monoclonal. Alternatively, the antibody is a polyclonal antibody. The preparation and use of polyclonal antibodies are also known the skilled artisan. The disclosure also encompasses hybrid antibodies, in which one pair of heavy and light chains is obtained from a first antibody, while the other pair of heavy and light chains is obtained from a different second antibody. Such hybrids may also be formed using humanized heavy and light chains. Such antibodies are often referred to as “chimeric” antibodies.

[0158] In general, intact antibodies are said to contain “Fc” and “Fab” regions. The Fc regions are involved in complement activation and are not involved in antigen binding. An antibody from which the Fc’ region has been enzymatically cleaved, or which has been produced without the Fc’ region, designated an “F(ab’)2” fragment, retains both of the antigen binding sites of the intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an “Fab1” fragment, retains one of the antigen binding sites of the intact antibody. Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain, denoted “Fd.” The Fd fragments are the major determinants of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity). Isolated Fd fragments retain the ability to bind specifically to immunogenic epitopes.

[0159] Methods of preparing antibodies are well known to those of ordinary skill in the science of immunology. Antibodies can be made by any of the methods known in the art utilizing a soluble polypeptide, or immunogenic fragment thereof, as an immunogen. One method of obtaining antibodies is to immunize suitable host animals with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production. The immunogen will facilitate presentation of the immunogen on the cell surface. Immunization of a suitable host can be carried out in a number of ways. Nucleic acid sequences encoding polypeptides or immunogenic fragments thereof, can be provided to the host in a delivery vehicle that is taken up by immune cells of the host. The cells will in turn express the polypeptide thereby generating an immunogenic response in the host. Alternatively, nucleic acid sequences encoding human polypeptides or immunogenic fragments thereof, can be expressed in cells in vitro, followed by isolation of the polypeptide and administration of the polypeptide to a suitable host in which antibodies are raised.

[0160] Alternatively, antibodies may, if desired, be derived from an antibody phage display library. A bacteriophage is capable of infecting and reproducing within bacteria, which can be engineered, when combined with human antibody genes, to display human antibody proteins. Phage display is the process by which the phage is made to 'display' the human antibody proteins on its surface. Genes from the human antibody gene libraries are inserted into a population of phage. Each phage carries the genes for a different antibody and thus displays a different antibody on its surface.

[0161] Antibodies made by any method known in the art can then be purified from the host. Antibody purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin.

[0162] Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods of making hybridomas are well known in the art. The hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source. Polynucleotides encoding the antibody of interest can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or recombinantly from these DNA sequences. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid. The method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse. The mammal may be primed for ascites production by prior administration of a suitable composition (e.g., Pristane).

[0163] Monoclonal antibodies (Mabs) produced by methods of the disclosure can be "humanized" by methods known in the art. "Humanized" antibodies are antibodies in which at least part of the sequence has been altered from its initial form to render it more like human immunoglobulins. Techniques to humanize antibodies are particularly useful when non-human animal (e.g., murine) antibodies are generated. Examples of methods for humanizing a murine antibody are provided in U.S. patents 4,816,567, 5,530,101, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.

[0164] Aptamers

[0165] In embodiments of the disclosure the capture molecule is an aptamer. Nucleic acid aptamers are single-stranded nucleic acid (DNA or RNA) ligands that function by folding into a specific globular structure that dictates binding to target proteins or other molecules with high affinity and specificity, as described by Osborne et al., Curr. Opin. Chem. Biol. 1:5-9, 1997; and Cerchia et al., FEBS Letters 528: 12-16, 2002. Desirably, the aptamers are small, approximately -15KD. The aptamers are isolated from libraries consisting of some 1014- 1015random oligonucleotide sequences by a procedure termed SELEX (systematic evolution of ligands by exponential enrichment). See Tuerk et al., Science, 249:505-510, 1990; Green et al., Methods Enzymology. 75-86, 1991;Gold et al., Annu. Rev. Biochem., 64: 763-797, 1995; Uphoff et al., Curr. Opin. Struct. Biol., 6: 281-288, 1996. Methods of generating aptamers are known in the art and are described, for example, in U.S. Patent No. 6,344,318, 6,331,398, 6,110,900, 5,817,785, 5,756,291, 5,696,249, 5,670,637, 5,637,461, 5,595,877, 5,527,894, 5,496,938, 5,475,096, 5,270,163, and in U.S. Patent Application Publication Nos. 20040241731, 20030198989, 20030157487, and 20020172962.

[0166] Therapeutic Proteins

[0167] Biomarkers of cardiotoxicity have a direct impact on clinical practice by enhancing cardiovascular risk stratification in patients receiving anthracyclines, facilitating early referrals to cardio-oncology specialty care, tailoring the use of cardioprotective therapies, and offering personalized cancer therapy options for patients. It was previously found that macrophages transport the heme-binding protein hemopexin to the heart to confer cardioprotection in mice treated with doxorubicin. Hemopexin facilitates lysosomal degradation of heme.

[0168] Accordingly, it is expected that the markers disclosed herein (e.g., SERPINA12, CPVL, PIGR, IQCF3, SESQ2, CSPG2, SHC2, and / or CFC1) also serve to provide cardioprotection to subjects when administered in therapeutic amounts.

[0169] Methods of Treatment

[0170] The present disclosure provides compositions and methods for treating subjects having or at risk of cardiotoxicity. In some embodiments, the subject has a neoplasia, e.g., a hematologic malignancy, and is need of an anthracycline treatment. A biologic sample is obtained from the subject and tested to detect levels of a biomarker described herein (e.g., CPVL, PIGR, or a biomarker of Table 1). Subjects having altered, e.g., increased, levels of such biomarkers relative to a reference are identified as having a propensity to develop cardiotoxicity, and may be selected to receive a cardioprotective agent prior to, concurrent with, or subsequent to anthracycline cancer therapy. In some embodiments, a subject identified as having a propensity to develop cardiotoxicity may be administered a reduced dose of anthracycline. In some embodiments, a subject identified as having a propensity to develop cardiotoxicity may be administered a cancer therapy that does not comprise an anthracycline. In some embodiments, a subject identified as having a propensity to develop cardiotoxicity may be administered a cancer therapy that comprises anthracycline and another cancer therapy agent.

[0171] In some embodiments, a subject is selected for a treatment when the biological sample of the subject has an increased binding in the capture molecule and the polypeptide biomarker (e.g., of Table 1) relative to a reference, thereby detecting cardiotoxicity or a propensity to develop cardiotoxicity in the subject. In some aspects, the disclosure provides panels comprising polypeptide biomarkers, e.g., a polypeptide as provided in Table 1, polynucleotides encoding polypeptide biomarkers of Table 1, or capture molecules that specifically bind such biomarkers, for characterizing the risk of cardiotoxicity in the subject prior to administration of an anthracycline. In some embodiments, elevated IgA levels in the subject are associated with increased risk for cardiotoxicity with anthracycline treatment.

[0172] A polypeptide biomarker may be detected in a biological sample of the subject, e.g., tissue or fluid, including, but not limited to, blood, blood serum, plasma, saliva, urine, ascites, cyst fluid, a homogenized tissue sample, a tissue sample obtained by biopsy, or a cell isolated from a patient sample.

[0173] In some aspects, a subject is identified as having a propensity to develop cardiotoxicity and is selected for a treatment when there is an alteration, e.g., an increase, in the level of one or more of the polypeptide or polynucleotide biomarkers of the patient’s biological sample relevant to a reference sample. The reference sample may be for example, a biological sample taken from a healthy subject, the patient themselves at a different time, or a subject who was observed not to develop cardiotoxicity after anthracycline treatment.

[0174] In some embodiments, a subject is identified as having a propensity to develop cardiotoxicity and is selected for a treatment when the increase in the level of the biomarker in a subject’s biological sample is at least 1.5 fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold more than the reference sample.

[0175] In some embodiments, a subject is identified as having a propensity to develop cardiotoxicity and is selected for a treatment when the decrease in the level of the biomarker in a subject’s biological sample is at least 1.5 fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold less than the reference sample.

[0176] In some embodiments, a patient identified by the methods presented herein as having a propensity to develop cardiotoxicity has a neoplasia, e.g., a hematologic malignancy, and is administered a reduced dosage of an anthracycline. In some embodiments, a selected patient having a neoplasia is administered a cancer therapy that does not comprise an anthracycline. In some embodiments, a selected patient having a neoplasia is administered a cancer therapy that is not cardiotoxic. In some embodiments, a selected patient having a neoplasia is administered an anthracycline in combination with another cancer therapy. In some embodiments, a selected patient having a neoplasia is administered a cancer therapy in combination with a cardioprotective agent. In some embodiments, the cardioprotective agent is an iron chelator, e.g., a bisdioxopiperazine, e.g., dexrazoxane, a statin, an aldosterone receptor antagonist (MRA), an angiotensin-converting enzyme inhibitor (ACEI), an angiotensin receptor blocker (ARB), or a beta-blocker, e.g., timolol, metoprolol, propranolol, bisoprolol, and carvedilol.

[0177] The present disclosure also provides methods of treating cardiotoxicity that comprise administering a therapeutically effective amount of a biomarker provided herein, or pharmaceutical compositions thereof, to a subject, e.g., a mammal, such as a human. Thus, in some embodiments, the disclosure provides a method of treating a subject at risk for cardiotoxicity by administering to the mammal a therapeutic amount of a therapeutic protein (e.g., SERPINA12, CPVL, PIGR, IQCF3, SESQ2, CSPG2, SHC2, and / or CFC1) or pharmaceutical compositions thereof, as described herein, sufficient to treat the cardiotoxicity, under conditions such that the cardiotoxicity is treated.

[0178] In some embodiments, the therapeutic protein (e.g., SERPINA12, CPVL, PIGR, IQCF3, SESQ2, CSPG2, SHC2, and / or CFC1) or a composition thereof, as described herein, is administered to a subject in a targeted manner. In some embodiments, the therapeutic protein (e.g., SERPINA12, CPVL, PIGR, IQCF3, SESQ2, CSPG2, SHC2, and / or CFC1) or a composition thereof, as described herein is directly administered to cardiomyocytes.

[0179] In some aspects of the invention, a subject may be identified for a heart failure treatment when the blood sample of the subject has altered, e.g., increased levels of one or more of the biomarkers described herein (e.g., in Table 1). In some embodiments, the subject identified for a heart failure treatment may have a neoplasia. In some embodiments, the subject identified for a heart failure treatment may be administered a beta-blockers, angiotensin-converting enzyme (ACE) inhibitor, angiotensin receptor blocker (ARB), Sodium-Glucose Cotransporter 2 (SGLT2) inhibitor, and / or a mineralicorticoid receptor antagonist.

[0180] Alternatively, a composition may be delivered systemically, such as by intravenous administration. Other modes of administration (parenteral, mucosal, implant, intraperitoneal, intradermal, transdermal, intramuscular, intravenous including infusion and / or bolus injection, and subcutaneous) are generally known in the art.

[0181] In some embodiments, therapeutic proteins are administered in a medium suitable for injection, such as phosphate buffered saline, into a subject.

[0182] Pharmaceutical Compositions

[0183] Compositions contemplated in the present disclosure include pharmaceutical compositions comprising a therapeutic protein (e.g., SERPINA12, CPVL, PIGR, IQCF3, SESQ2, CSPG2, SHC2, and / or CFC1).

[0184] In other embodiments, the disclosure provides a formulation comprising a cancer therapy (e.g., anthracycline) and a cardioprotective agent (e.g., a bisdioxopiperazine, e.g., dexrazoxane, a statin, an aldosterone receptor antagonist (MRA), an angiotensin-converting enzyme inhibitor (ACEI), an angiotensin receptor blocker (ARB), or a beta-blocker (e.g., timolol, metoprolol, propranolol, bisoprolol, and carvedilol).

[0185] The therapeutic protein (e.g., SERPINA12, CPVL, PIGR, IQCF3, SESQ2, CSPG2, SHC2, and / or CFC1), as described herein, can be administered as a therapeutic composition (e.g., as pharmaceutical compositions). For example, compositions as described herein can be provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. A liquid preparation may be easier to prepare than a gel, another viscous composition, or a solid composition. Additionally, a liquid composition may be more convenient to administer (i.e., by injection). Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise a carrier, which can be a solvent or dispersing medium comprising, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

[0186] Sterile injectable solutions can be prepared by incorporating the therapeutic protein (e.g., SERPINA12, CPVL, PIGR, IQCF3, SESQ2, CSPG2, SHC2, and / or CFC1) in a sufficient amount of an appropriate diluent. Such compositions may be in admixture with a suitable carrier or excipient such as sterile water, physiological saline, glucose, dextrose, or another carrier or excipient suitable for delivering a therapeutic protein to a subject. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “Remington's Pharmaceutical Science,” 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0187] Additives that enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by an antibacterial or antifungal agent including, but not limited to, parabens, chlorobutanol, phenol, and sorbic acid. According to the present disclosure, however, any vehicle, diluent, or additive used must be compatible with the therapeutic protein. The compositions can be isotonic, i.e., they have the same osmotic pressure as blood and cerebrospinal fluid. The desired isotonicity of the compositions of this disclosure may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride may be suitable for buffers containing sodium ions.

[0188] Viscosity of the compositions, if desired, can be maintained at a selected level using a pharmaceutically acceptable thickening agent. In some embodiments, the thickening agent is methylcellulose, which is readily and economically available and is easy to work with. Other suitable thickening agents include, but are not limited to, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, and carbomer. The concentration of the thickener will depend upon the agent selected and the amount of the agent used. Suitable carriers and other additives may be chosen depending on the route of administration and the nature of the dosage form (e.g., a liquid dosage form can be formulated into a solution, a suspension, a gel, or another liquid form, such as a time release formulation or liquid-filled form).

[0189] The skilled artisan can readily determine the amounts of the active ingredient, and optional additives, vehicles, and / or carrier in compositions to be administered. In one embodiment any additive is present in an amount of about 0.001% to about 50 % (weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001% to about 5 wt %. In another embodiment, the active ingredient is present at about 0.0001% to about 1 wt %. In yet another embodiment, the active ingredient is present at about 0.0001% to about 0.05 wt %. In still other embodiments, the active ingredient is present at about 0.001% to about 20 wt %. In some embodiments, the active ingredient is present at about 0.01% to about 10 wt %. In another embodiment, the active ingredient is present at about 0.05% to about 5 wt %. For any composition to be administered to an animal or human, and for any particular method of administration, toxicity can be determined by measuring the lethal dose (LD) and LDso in a suitable animal model, e.g., a rodent such as mouse. The dosage of the composition(s), concentration of components therein, and timing of administering the composition(s), which elicit a suitable response can also be determined. Such determinations do not require undue experimentation in light of the knowledge of the skilled artisan, this disclosure, and the documents cited herein. The time for sequential administrations can also be ascertained without undue experimentation. The present disclosure further includes pharmaceutical compositions comprising a therapeutic protein (e.g., SERPINA12, CPVL, PIGR, IQCF3, SESQ2, CSPG2, SHC2, and / or CFC1), produced according to methods described herein. The compositions of the disclosure may comprise one or more polypeptides, as described herein, formulated in pharmaceutically- acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions of the disclosure may be administered in combination with other agents as well, such as, e.g., other proteins, polypeptides, small molecules or various pharmaceutically-active agents (e.g., anthracy clines). There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended gene therapy.

[0190] In the pharmaceutical compositions of the disclosure, formulation of pharmaceutically- acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g, oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation.

[0191] In certain aspects, the present disclosure provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more polypeptides, as described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., pharmaceutically acceptable protein buffer solutions).

[0192] Particular embodiments of the disclosure may comprise other formulations, such as those that are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000.

[0193] Hardware and Software

[0194] The disclosure also provides for the communication of assay results relating to cardiotoxicity or diagnoses or both to technicians, physicians or patients, for example. In certain embodiments, computers will be used to communicate assay results or diagnoses or both to interested parties, e.g, physicians and their patients. In some embodiments, the assays will be performed, or the assay results analyzed in a country or jurisdiction which differs from the country or jurisdiction to which the results or diagnoses are communicated.

[0195] The any of the methods described herein, the step of correlating the measurement of the biomarker(s) with cardiotoxicity (e.g., cardiotoxicity associated with anthracy cline treatment) in a subject (e.g., a subject having a hematologic malignancy) can be performed on general-purpose or specially-programmed hardware or software.

[0196] In aspects, the analysis is performed by a software classification algorithm. The analysis of analytes by any detection method well known in the art, including, but not limited to the methods described herein, will generate results that are subject to data processing. Data processing can be performed by the software classification algorithm. Such software classification algorithms are well known in the art and one of ordinary skill can readily select and use the appropriate software to analyze the results obtained from a specific detection method.

[0197] In aspects, the analysis is performed by a computer-readable medium. The computer- readable medium can be non- transitory and / or tangible. For example, the computer readable medium can be volatile memory (e.g., random access memory and the like) or non-volatile memory (e.g., read-only memory, hard disks, floppy discs, magnetic tape, optical discs, paper table, punch cards, and the like).

[0198] For example, analysis of analytes by time-of-flight mass spectrometry generates a time- of-flight spectrum. The time-of-flight spectrum ultimately analyzed typically does not represent the signal from a single pulse of ionizing energy against a sample, but rather the sum of signals from a number of pulses. This reduces noise and increases dynamic range. This time-of-flight data is then subject to data processing. Exemplary software includes, but is not limited to, Ciphergen’s ProteinChip® software, in which data processing typically includes TOF-to-M / Z transformation to generate a mass spectrum, baseline subtraction to eliminate instrument offsets and high frequency noise filtering to reduce high frequency noise.

[0199] Data generated by desorption and detection of biomarkers can be analyzed with the use of a programmable digital computer. The computer program analyzes the data to indicate the number of biomarkers detected, and optionally the strength of the signal and the determined molecular mass for each biomarker detected. Data analysis can include steps of determining signal strength of a biomarker and removing data deviating from a predetermined statistical distribution. For example, the observed peaks can be normalized, by calculating the height of each peak relative to some reference. The reference can be background noise generated by the instrument and chemicals such as the energy absorbing molecule which is set at zero in the scale.

[0200] The computer can transform the resulting data into various formats for display. The standard spectrum can be displayed, but in one useful format only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling biomarkers with nearly identical molecular weights to be more easily seen. In another useful format, two or more spectra are compared, conveniently highlighting unique biomarkers and biomarkers that are up- or down-regulated between samples. Using any of these formats, one can readily determine whether a particular biomarker is present in a sample.

[0201] Analysis generally involves the identification of peaks in the spectrum that represent signal from an analyte. Peak selection can be done visually, but software is available, for example, as part of Ciphergen’s ProteinChip® software package, that can automate the detection of peaks. This software functions by identifying signals having a signal-to-noise ratio above a selected threshold and labeling the mass of the peak at the centroid of the peak signal. In embodiments, many spectra are compared to identify identical peaks present in some selected percentage of the mass spectra. One version of this software clusters all peaks appearing in the various spectra within a defined mass range and assigns a mass (N / Z) to all the peaks that are near the mid-point of the mass (M / Z) cluster.

[0202] In aspects, software used to analyze the data can include code that applies an algorithm to the analysis of the results (e.g., signal to determine whether the signal represents a peak in a signal that corresponds to a biomarker according to the present disclosure). The software also can subject the data regarding observed biomarker peaks to classification tree or ANN analysis, to determine whether a biomarker peak or combination of biomarker peaks is present that indicates the status of the particular clinical parameter under examination. Analysis of the data may be “keyed” to a variety of parameters that are obtained, either directly or indirectly, from the mass spectrometric analysis of the sample. These parameters include, but are not limited to, the presence or absence of one or more peaks, the shape of a peak or group of peaks, the height of one or more peaks, the log of the height of one or more peaks, and other arithmetic manipulations of peak height data. Classification Algorithms

[0203] In some embodiments, data derived from the assays (e.g., ELISA assays) that are generated using samples such as “known samples” can then be used to “train” a classification model. A “known sample” is a sample that has been pre-classified. The data that are derived from the spectra and are used to form the classification model can be referred to as a “training data set.” Once trained, the classification model can recognize patterns in data derived from spectra generated using unknown samples. The classification model can then be used to classify the unknown samples into classes. This can be useful, for example, in predicting whether or not a particular biological sample is associated with a certain biological condition (e.g., susceptibility to cardiotoxicity vs. non-susceptibility to cardiotoxicity associated with anthracycline treatment in a subject having a hematologic malignancy).

[0204] The training data set that is used to form the classification model may comprise raw data or pre-processed data. In some embodiments, raw data can be obtained directly from time-of- flight spectra or mass spectra, and then may be optionally “pre-processed” as described above.

[0205] Classification models can be formed using any suitable statistical classification (or “learning”) method that attempts to segregate bodies of data into classes based on objective parameters present in the data. Classification methods may be either supervised or unsupervised. Examples of supervised and unsupervised classification processes are described in Jain, “Statistical Pattern Recognition: A Review”, IEEE Transactions on Pattern Analysis and Machine Intelligence, Vol. 22, No. 1, January 2000, the teachings of which are incorporated by reference.

[0206] In supervised classification, training data containing examples of known categories are presented to a learning mechanism, which learns one or more sets of relationships that define each of the known classes. New data may then be applied to the learning mechanism, which then classifies the new data using the learned relationships. Examples of supervised classification processes include linear regression processes (e.g., multiple linear regression (MLR), partial least squares (PLS) regression and principal components regression (PCR)), binary decision trees (e.g., recursive partitioning processes such as CART - classification and regression trees), artificial neural networks such as back propagation networks, discriminant analyses (e.g., Bayesian classifier or Fischer analysis), logistic classifiers, and support vector classifiers (support vector machines). In embodiments, a supervised classification method is a recursive partitioning process. Recursive partitioning processes use recursive partitioning trees to classify spectra derived from unknown samples. Further details about recursive partitioning processes are provided in U.S. Patent Application No. 2002 0138208 Al to Paulse etal., “Method for analyzing mass spectra.”

[0207] In other embodiments, the classification models that are created can be formed using unsupervised learning methods. Unsupervised classification attempts to learn classifications based on similarities in the training data set, without pre-classifying the spectra from which the training data set was derived. Unsupervised learning methods include cluster analyses. A cluster analysis attempts to divide the data into “clusters” or groups that ideally should have members that are very similar to each other, and very dissimilar to members of other clusters. Similarity is then measured using some distance metric, which measures the distance between data items, and clusters together data items that are closer to each other. Clustering techniques include the MacQueen’s K- means algorithm and the Kohonen’s Self-Organizing Map algorithm.

[0208] Learning algorithms asserted for use in classifying biological information are described, for example, in PCT International Publication No. WO 01 / 31580 (Barnhill et al. , “Methods and devices for identifying patterns in biological systems and methods of use thereof’), U.S. Patent Application No. 2002 0193950 Al (Gavin et al., “Method or analyzing mass spectra”), U.S. Patent Application No. 2003 0004402 Al (Hitt etal., “Process for discriminating between biological states based on hidden patterns from biological data”), and U.S. Patent Application No. 2003 0055615 Al (Zhang and Zhang, “Systems and methods for processing biological expression data”).

[0209] The classification models can be formed on and used on any suitable digital computer. Suitable digital computers include micro, mini, or large computers using any standard or specialized operating system, such as a Unix, Windows™ or Linux™ based operating system. The digital computer that is used may be physically separate from the mass spectrometer that is used to create the spectra of interest, or it may be coupled to the mass spectrometer.

[0210] The training data set and the classification models according to embodiments of the disclosure can be embodied by computer code that is executed or used by a digital computer. The computer code can be stored on any suitable computer readable media including optical or magnetic disks, sticks, tapes, etc., and can be written in any suitable computer programming language including C, C++, visual basic, etc. The learning algorithms described above are useful both for developing classification algorithms for the biomarkers already discovered, or for finding new biomarkers for susceptibility to cardiotoxicity associated with anthracy cline treatment in a subject having a hematologic malignancy. The classification algorithms, in turn, form the base for diagnostic tests by providing diagnostic values (e.g., cut-off points) for biomarkers used singly or in combination.

[0211] Kits

[0212] Provided herein are kits for the diagnosis, treatment, and / or prevention of cardiotoxicity associated with anthracycline treatment in a subject, e.g., a subject having a hematologic malignancy. In one embodiment, the kit includes a capture reagent, e.g., an antibody or nucleic acid molecule, that binds specifically to a biomarker disclosed herein, e.g., as provided in Table 1.

[0213] In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic cellular composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0214] If desired an agent of the disclosure is provided together with instructions for administering the agent to a subject having or at risk of developing cardiotoxicity associated with anthracycline treatment in a subject having a hematologic malignancy. The instructions will generally include information about the use of the composition for the treatment or prevention of the disease or disorder. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of cardiotoxicity associated with anthracycline treatment in a subject having a hematologic malignancy or symptoms thereof; precautions; warnings; indications; counterindications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing the disclosure. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0215] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.

[0216] EXAMPLES

[0217] Example 1: Identification of Biomarkers of Cardiotoxicity in Patients with Hematologic Malignancies Prior to Treatment with an An thracy cline

[0218] An aptamer-based proteomics platform was used to measure over 7000 proteins in plasma obtained from a prospectively enrolled cohort of patients with hematologic malignancies (n=33). Samples were available at baseline and 3 months after the initiation of anthracyclines- based cancer therapy. Echocardiography was performed at baseline and 6 to 12 months after initiation of anthracy clines. Pearson’s correlation was used to compare normalized baseline protein levels to the change in either global longitudinal strain (AGLS) or left ventricular ejection fraction (ALVEF) adjudicated by a cardiologist blinded to patient identity and time point.

[0219] The cohort consisted of 18 men and 16 women with an average age of 60 ± 14.7 years (Table 2 below). The average relative ALVEF was -0.11 ± 0.14%, and the average relative AGLS was -0.11 ± 0.19%. In this cohort, several proteins with baseline levels that correlated with AGLS and / or ALVEF were identified using a false discovery rate (FDR) threshold of q<0.05. These markers included SERPINA12 (serine protease inhibitor and adipokine; R= -0.73 for AGLS and -0.65 for ALVEF); CPVL (carboxypeptidase implicated in macrophage lysosomal activity; R= -0.69 for AGLS and -0.79 for ALVEF), PIGR (IgA / IgM receptor; R= -0.54 for AGLS and -0.74 for ALVEF), IQCF3 (predicted to enable calmodulin binding; R= -0.62 for AGLS and -0.72 for ALVEF), and SESQ2 (required for endocytic trafficking; R= -0.60 for AGLS and -0.70 for ALVEF). These findings remained significant after adjusting for age, sex, race, body mass index (BMI), and estimated glomerular filtration rate (eGFR). Additional markers shown to corelate with ALVEF include Versican core protein (CSPG2), SHC- transforming protein 2 (SHC2), and Cryptic protein (CFC1). The results are summarized in Tables 3 and 4below.

[0220] Table 2: Demographic Characteristics of Cohort

[0221]

[0222] Table 3: Echocardiographic Parameters

[0223] Table 4: Correlations Between Biomarkers and Cardiac Function

[0224] In sum, in this cohort, an association between pre-anthracyclines levels of specific plasma proteins and subsequent decline in cardiac function was observed, as assessed by echocardiography.

[0225] This study identified new polypeptide biomarkers of cardiotoxicity in patients with hematologic malignancies treated with anthracyclines. New polypeptide biomarkers of cardiotoxicity have a direct impact on clinical practice by enhancing cardiovascular risk stratification in patients receiving anthracyclines, facilitating early referrals to cardio- oncology specialty care, tailoring the use of cardioprotective therapies, and offering personalized cancer therapy options for patients. It was previously found that macrophages transport the hemebinding protein hemopexin to the heart to confer cardioprotection in mice treated with doxorubicin. Hemopexin facilitates lysosomal degradation of heme. Without intending to be bound by theory, CPVL, as a macrophage lysosomal enzyme, was found to represent another member of this pathway that can be developed into a polypeptide biomarker or therapeutic for use in patients at risk for anthracyclines cardiomyopathy.

[0226] Example 2: The Immunologic Markers PIGR and CPVL Correlate with Anthracycline- Associated Cardiac Dysfunction

[0227] The following Example provide additional results in follow up to the clinical study described in Example 1, to identify plasma proteins that correlate with the development of anthracycline cardiotoxicity. Validation studies in preclinical mouse models were also performed. The aptamer-based proteomics platform described in Example 1 was used to measure over 7000 proteins in plasma obtained from a prospectively enrolled cohort of patients with hematologic malignancies at baseline and 3 months after anthracycline cancer therapy (n=34). Transthoracic echocardiography was used to correlate change in left ventricular ejection fraction (ALVEF) with protein levels. Candidate polypeptide biomarkers were validated in a second patient cohort treated with anthracyclines for hematologic malignancies (n=28) as well as in a mouse model of delayed doxorubicin (Dox) cardiotoxicity.

[0228] In both discovery and validation cohorts, baseline levels of carboxypeptidase vitellogenic-like protein (CPVL) and poly-immunoglobulin receptor (PIGR) measured in the plasma prior to anthracyclines correlated with a subsequent decline in LVEF. Findings were confirmed in human plasma samples using enzyme-linked immunosorbent assay (ELISA) or by western blot, and similar changes in proteins were observed in mice treated with Dox. In further support of PIGR as a mechanistic polypeptide biomarker representing a systemic adaptive immune response, Dox treatment was associated with increased circulating levels of IgA+ B- cells in mice, which similarly correlated with the decline in LVEF.

[0229] Pre-anthracycline levels of the plasma immunologic proteins CPVL and PIGR were associated with a subsequent decline in cardiac function in both mice and humans treated with anthracyclines, representing potentially new inflammatory polypeptide biomarkers of anthracycline cardiotoxicity.

[0230] Methods

[0231] Study Design and Patient Enrollment

[0232] Eligible participants were individuals aged 18 years or older with hematologic malignancies who were scheduled to receive treatment with anthracyclines. Participants additionally had at least one risk factor for the development of cardiotoxicity, including: age greater than 65 years, hypertension, treatment for aggressive non-Hodgkin’s lymphoma, prior chest irradiation, or a planned cumulative doxorubicin dose > 300 mg / m2or patients receiving anthracyclines other than doxorubicin, conversions were made using an equivalent doxorubicin dose based on prior publications.

[0233] Patients were excluded if they had a baseline left ventricular ejection fraction (LVEF) of less than 50% or underwent an initial blood draw but did not proceed with the planned cancer therapy. Participants underwent a baseline transthoracic echocardiogram before receiving the initial dose of anthracycline cancer therapy. A baseline blood draw was obtained along with answers to a questionnaire assessing cardiovascular risk factors. A follow-up echocardiogram was performed at 12 (± 6) months. Left ventricular ejection fraction (LVEF) and global longitudinal strain (GLS) were measured by an experienced cardiologist blinded to patient identification and study time points. Heart failure was adjudicated by manual chart review according to published criteria, as our group has previously published. A study timeline is depicted in FIG. 1.

[0234] Mouse Doxorubicin Model

[0235] For proteomics experiments, male C57BL / 6 mice at 12 weeks of age were treated with doxorubicin 3 mg / kg (Tocris Bioscience, 25316-40-9) or the corresponding vehicle (saline), administered by intraperitoneal injection every 2 days for 2 weeks (total of eight injections). Where indicated, male C57BL / 6 mice at 8-12 weeks of age were dosed with doxorubicin 5 mg / kg doxorubicin or saline by intraperitoneal injection weekly for 4 or 8 weeks. A minimum sample size of 6 mice per group was calculated to detect statistically significant changes in cardiac function with a power of greater than 80% and a= 0.05. All analyses were performed in a blinded fashion using an identification number assigned by a third party.

[0236] Aptamer-Based Proteomics

[0237] In patients, baseline and post-anthracycline cancer therapy plasma samples were analyzed using SomaScan®, an aptamer-based proteomics platform to measure 7288 proteins, according to the manufacturer’s protocol.

[0238] Mouse Echocardiography

[0239] Transthoracic echocardiography was performed 4-8 weeks after the first Dox dose on the day before tissue collection. Mice were sedated using isoflurane on a heated stage in supine position. Heart rate and respiratory rate were continuously monitored via stage electrodes. Depilatory cream (Nair) was applied to the chest to remove fur, and ultrasonic gel was applied to a 22-55 MHz echocardiography transducer (MS550D Vevo 2100, Fujifilm VisualSonics) to obtain parasternal short axis views of the left ventricle at the level of papillary muscles in M- mode with a target heart rate of 400-500 bpm. Cardiac parameters and heart rate were measured by averaging the values from five cardiac cycles. All analyses were performed by a blinded observer using Vevo 2100 software version 2.2.0 (Build 12089).

[0240] ELISAs Human or mouse plasma samples were used for the following ELISAs at the following dilutions, all according to the manufacturer’s instructions: human PIGR (Abeam 282302 - 1 :250), human IgA (Invitrogen 88-50-600, 1 : 10,000), mouse PIGR (LS Bio LS-F7408, 1 :25).

[0241] Flow Cytometry

[0242] Hearts were excised from mice and perfused with PBS, then minced and digested enzymatically with 0.895 mg ml-^ collagenase II (Gibco, 17101-015). Single-cell suspensions were generated from cells in culture or lymphoid organs using a 40-pm cell strainer. Blood samples first underwent red blood cell (RBC) lysis using RBC lysis buffer (Biolegend, 420302) according to the manufacturer’s instructions, then leukocytes were pelleted before staining. Samples were stained using the following antibodies: CD45 FITC (Biolegend 109806), CD45 BV421 (Biolegend 109832), CD3 APC Cy7 (Biolegend 100330), CD19 BV711 (Biolegend 115555), or IgA PE (Thermofisher 12-4204-82), alongside mouse Fc block (Biolegend 101320). Samples were surface stained by incubation with the relevant antibodies listed above diluted 1 : 100, or algA 1 : 50 in PBS + 2% fetal bovine serum (FBS) for 20 min at 4 °C. Data were acquired on an Attune CytPix Flow Cytometer (Thermo Fisher) and analyzed using FlowJo software Version 10.10.0.

[0243] Immunoblotting

[0244] Plasma samples were diluted 1:10 in 50 mM Tris pH 7.1 with 150 mM NaCl, and 0.5% NP-40, then incubated at 4°C for 1 hour with protein A / G beads to remove Ig (Santa Cruz Biotechnologies 2003). Cardiac samples were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche, 4906845001 and 4693159001) then cleared by centrifugation at 4 °C at 13,000g. Protein concentrations were determined using DC protein assay (Bio-Rad, 5000112) and equal amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis, then transferred to polyvinylidene difluoride membranes, followed by blocking with Tris-buffered saline + 0.1% Tween20 containing 5% BSA or non-fat milk for 1 hour at room temperature (RT). Immunoblotting was performed overnight at 4 °C using antibodies diluted 1: 1000. Antibody sources were as follows: CPVL (Abeam 180147), GAPDH (CST 2118), and P-actin(CST 4970). Blots were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:2,000: rabbit (CST 7074) at RT for 1 h, then developed using Pierce ECL (Thermo Fisher 32106) and imaged using a ChemiDoc XRS+ System (Bio-Rad). Band intensity was quantified using Imaged.

[0245] THP-1 Cell Derived Macrophage Culture

[0246] Thpl cells were cultured in suspension in RPMI medium (Gibco 11875-093) containing 10% fetal bovine serum (FBS - Atlanta Biologicals SI 1150), Glutamax (Gibco A12860-01), NaHCCh (Gibco 25080-094), Penicillin / Streptomycin (Gibco 15140122), 60pM P- mercaptoethanol (Sigma 444203), and Sodium pyruvate (Gibco 11360-070). Cells were plated in 6 well dishes and treated for 48 hours with 200 ng / mL PMA (Sigma P8139) and 100 ng / mL LPS (Invitrogen 103576-100) before treatment with Dox.

[0247] Mouse Bone Marrow -Derived Macrophage Culture

[0248] Bone marrow was isolated from mice aged 6-12 weeks old, filtered through a 100 pM cell strainer, followed by red blood cell lysis was performed according to manufacturer’s instructions (Biolegend 4210301). Bone marrow cells were plated at a concentration of 1 x 107cells in a 10 cm dish and cultured in RPBI medium containing 10% FBS, Glutamax, Penicillin / Streptomycin, 60pM P-mercaptoethanol, Sodium pyruvate, and 20% L929 cell conditioned media.

[0249] Mouse CD8+T-cell Culture

[0250] CD8+T-cells were isolated from splenic cell suspensions by positive selection using magnetic beads (Miltenyi or 130-117-044). Cells were plated at 2 million cells / mL and activated in the presence of anti-CD3 antibody (2.5 pg / mL, Biolegend 100253) and anti-CD28 antibody (1 pg / mL, Biolegend 102102), IL-12 (10 ng / mL, Peprotech 210-12) and, anti-IL4 (50 ng / mL, Biolegend 504102) for 3 days then harvested for experiments. T-cells were dosed with the indicated concentration of DR where indicated. T-cells were cultured at 37°C in RPMI medium (Gibco 11875-093) containing 10% fetal bovine serum (FBS - Atlanta Biologicals SI 1150), Glutamax (Gibco A12860-01), NaHCCh (Gibco 25080-094), Penicillin / Streptomycin (Gibco 15140122), 60pM P-mercaptoethanol (Sigma 444203), and Sodium pyruvate (Gibco 11360-070).

[0251] Statistical Analysis For aptamer-based proteomics, relative fluorescent units were log normalized. In patients, Pearson’s correlation was used to compare baseline protein levels to the change in left ventricular ejection fraction (ALVEF). Statistical significance was determined using a Benjamini-Hochberg false discovery rate (FDR) q<0.05. Multivariable adjustment was performed for proteins that met statistical significance based on the FDR q value. Multivariable models were adjusted for clinical characteristics that could potentially affect protein levels, such as age, sex, body mass index (BMI), and estimated glomerular filtration rate (eGFR) using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. In multivariable- adjusted models, we used a p-value <0.05 to declare statistical significance. Statistical analyses were performed using STATA 14® or Prism 10®. In mice, saline and Dox-treated groups were compared using an unpaired t-test or 1-way ANOVA with Tukey’s multiple comparison test. Statistical significance was considered at a two-tailed alpha <0.05.

[0252] Results

[0253] The discovery cohort consisted of 34 patients with hematologic malignancies enrolled prospectively from 2018 to 2022 (FIG. 1). The patients received an average of 276.9 ± 57.4 mg / m2of doxorubicin or equivalent anthracycline. On average, the LVEF declined by 0.11 ± 0.14%. Only one patient developed heart failure. Additional patient characteristics can be found in Table 5 below.

[0254] Table 5: Patient Characteristics

[0255] Using aptamer-based proteomics to measure 7288 proteins, baseline (pre-cancer therapy) levels of 24 proteins were found to correlate with ALVEF using a false discovery rate (FDR) threshold of q<0.05 (Table 6, below). These included several proteins with immunologic functions, such as the poly-immunoglobulin receptor (PIGR), carboxypeptidase vitellogenic-like protein (CPVL), and tyrosine-protein phosphatase non-receptor type 2 (TCPTP).

[0256] Table 6: Human Proteomics Results

[0257] The top two proteins were most highly correlated with ALVEF, PIGR (R=-0.74, p = 0.0022) and CPVL (R=-0.74, p = 0.0022). Both associations remained significant after adjusting for clinical covariates including age, sex, body mass index (BMI), and eGFR (Tables 7 and 8, below). Given that the patient who experienced heart failure was an outlier in both analyses, this patient was excluded. However, the associations between baseline PIGR and CPVL persisted (FIGS. 5 A and 5B).

[0258] Table 7: Adjustment of Top Hits for Confounding Factors in Human Proteomics

[0259] Table 8: Adjustment of Top Hits for Confounding Factors in Human Proteomics

[0260] The PIGR-IgA Axis Is Associated with Early Cardiotoxicity in Anthracycline-Treated Patients and Mice

[0261] The top protein associated with ALVEF in anthracycline-treated patients (FIG. 2A) was polymeric immunoglobulin receptor (PIGR). This relationship was measured in the discovery cohort using a commercially available ELISA (FIG. 2B). The relationship between pre-treatment PIGR levels and ALVEF in a second validation cohort enrolled was also performed (n = 28). Similar correlation between PIGR and ALVEF following anthracy cline treatment were observed in both the discovery and validation cohorts, as shown in FIG. 2C.

[0262] As PIGR mediates the secretion of mucosal immunoglobulin across endothelial and epithelial surfaces, the levels of circulating immunoglobulin were also assessed. Using a commercially available IgA ELISA, it was found that circulating IgA correlated with ALVEF in both discovery and validation cohorts (FIGS. 2D-2E). PIGR and IgA were also robustly correlated with each other (FIG. 2F). Taken together, these data indicate PIGR-mediated secretion of IgA is associated with early anthracycline cardiotoxicity. Next, wild type mice were administered either Dox 5 mg / kg intraperitoneally (i.p. ,) or saline, weekly for 4 or 8 weeks. A progressive decline in fractional shortening and increased dilation of the left ventricle were observed (FIGS. 3A-3C). PIGR levels correlated with fractional shortening (R=-0.77, p=0.07) and left ventricular diameter (R=0.89, p=0.015) in mice treated with Dox (FIGS. 3D-3F).

[0263] Given the finding that plasma IgA correlated with ALVEF in patients treated with anthracyclines, CD19+B-cells and IgA+CD19+B-cells were measured in the circulation of mice using flow cytometry. The gating strategy for these experiments is shown in FIG. 6A. Overall circulating CD19+B-cells transiently decreased after 4 weeks of Dox treatment and then returned to baseline after 8 weeks (FIG. 3G). However, in contrast, circulating CD 19+IgA+B-cells were increased after 8 weeks of Dox (FIG. 3H). Notably, the number of CD 19+IgA+B-cells correlated with fractional shortening across all groups of mice (Rvalue = -0.5519, R2=0.3046, and p value=0.0142). Changes in cardiac tissue B-cells (FIG. 6B) or IgA+B-cells (FIG. 6C) were not observed, suggesting that the IgA+B-cell response induced by Dox is systemic rather than cardiac-specific in mice (FIGS. 5B and 5C). Together, these data demonstrate a PIGR- immunoglobulin axis in patients and in rodent models of anthracycline cardiotoxicity.

[0264] Circulating CPVL Is Associated with Early Cardiotoxicity in Anthracy cline-Treated Patients and Mice

[0265] After CPVL was observed to correlate with ALVEF in the discovery cohort (FIG. 4A), confirmation in the validation cohort was also investigated. As no commercially available ELISA kits or published quantitative methods were available for human CPVL, the proteomics results were assessed via immunoblot on patient plasma. As this technique is not high- throughput, baseline plasma CPVL levels in patients were compared that had either no change in LVEF to patients that had a greater than 20% decline in LVEF. Higher CPVL levels were observed in patients with a greater than 20% decline in LVEF (FIGS. 4B and 4D). A similar analysis was then performed on samples from the validation cohort and higher circulating CPVL in patients were found with a greater than 20% drop in LVEF (FIGS. 4C and 4E), confirming that higher circulating CPVL levels prior to anthracycline treatment correlate with decreased systolic function after anthracycline treatment. To assess whether this pathway could be further studied in mouse models, lysates from mouse bone marrow derived macrophages (BMDMs), which should express high levels of CPVL, were compared to CD8+T-cell lysates, which are not known to express CPVL. Expression of CPVL in macrophage but not T-cell lysates was observed (FIG. 7). As macrophages and monocytes are the main cell type known to express CPVL, it was next assessed whether doxorubicin treatment induces CPVL expression or secretion. Thpl derived macrophages were generated and treated with increasing doses of Dox. No change in CPVL expression in the lysates was observed, and CPVL was undetectable in the supernatant (FIG. 8). This antibody was used to compare CPVL levels in mice treated with saline or 8 weeks of Dox and an increase in plasma CPVL was observed (FIGS. 4F and 4H) but not CPVL in whole cardiac lysate (FIGS. 4G and 41). Circulating levels of CPVL, as determined by immunoblot of plasma samples, correlated with fractional shortening in saline and 8 week Dox-treated mice (FIG. 4J). Together, these data indicate that Dox also induces circulating CPVL that correlates with the development of systolic dysfunction.

[0266] Other Embodiments

[0267] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0268] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0269] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A panel for characterizing cardiotoxicity in a biological sample of a subject, the panel comprising capture molecules that specifically bind one or more polypeptide biomarkers selected from the group consisting of: carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase non-receptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF- 16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain- containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7, or a polynucleotide encoding the polypeptide biomarker.

2. The panel of claim 1, wherein the one or more polypeptide biomarkers are carboxypeptidase vitellogenic like (CPVL) and / or polymeric immunoglobulin receptor (PIGR).

3. The panel of claim 1 or claim 2, wherein the capture molecule is an aptamer, a polynucleotide probe, an antibody, or antigen binding fragment thereof.

4. The panel of claim 3, wherein the capture molecule is an antibody.

5. The panel of any one of claims 1-4, wherein the capture molecule is bound to a substrate.

6. The panel of claim 5, wherein the substrate is a chip, bead, microfluidic platform, or membrane.

7. A method of characterizing cardiotoxicity or a propensity to develop cardiotoxicity in a subject, the method comprising(a) contacting a biological sample of the subject with the panel of any one of claims 1-6; and(b) detecting an increase in binding of the capture molecule and the polypeptide biomarker relative to a reference, thereby detecting cardiotoxicity or a propensity to develop cardiotoxicity in the subject.

8. The method of claim 7, wherein the method further comprises measuring IgA levels in the biological sample.

9. The method of claim 7 or claim 8, wherein the biological sample is a blood sample.

10. The method of claim 7 or claim 8, wherein the biological sample is a plasma or serum sample.

11. The method of any one of claims 7-10, wherein the subject has not been treated with an anthracycline.

12. The method of any one of claims 7-10, wherein the subject has been treated with an anthracycline.

13. A method of treating a selected subject having a neoplasia, comprising administering a reduced dosage of a treatment comprising anthracyclines to the subject, wherein the subject is selected by characterizing a biological sample of the subject as having an alteration in the level of a polypeptide or polynucleotide polypeptide biomarker relative to a reference, wherein the polypeptide biomarker is selected from the group consisting of carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase non-receptor type 2 (TCPTP),Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain-containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7, or a polynucleotide encoding the polypeptide biomarker.

14. The method of claim 13, wherein the polypeptide biomarkers are CPVL and / or PIGR.

15. A method of treating a selected subject having a neoplasia, comprising administering a cancer therapy agent to the selected subject, wherein the subject is selected by characterizing a biological sample of the subject as having an alteration in the level of a polypeptide biomarker relative to a reference, wherein the polypeptide biomarker is selected from the group consisting carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC- transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase nonreceptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain- containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7, or a polynucleotide encoding the polypeptide biomarker, and wherein the cancer therapy agent does not comprise an anthracycline.

16. The method of claim 15, wherein the polypeptide biomarkers are CPVL and / or PIGR.

17. A method of treating a selected subject having a neoplasia, comprising administering a cancer therapy agent to the subject in combination with a cardioprotective agent, wherein the subject is selected by characterizing a biological sample of the subject as having an alteration in the level of a polypeptide biomarker relative to a reference, wherein the biomarker is selected from the group consisting carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase non-receptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF- 16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain- containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), Carbonic anhydrase 7, and polynucleotides encoding the polypeptides .

18. The method of claim 17, wherein the biomarkers are CPVL and / or PIGR.

19. The method of claim 17 or claim 18, wherein the cancer therapy comprises an anthracycline or a pharmaceutically acceptable salt thereof.

20. The method of any one of claims 17-19, wherein the cardioprotective agent comprises one or more of: an iron chelator, a statin, an aldosterone receptor antagonist (MRA), an angiotensin-converting enzyme inhibitor (ACEI), an angiotensin receptor blocker (ARB), or a beta-blocker.

21. The method of any one of claims 13-20, wherein the level of the polypeptide or polynucleotide biomarker is increased relative to the reference.

22. A method of treating a subject having a neoplasia, the method comprising administering a treatment comprising an anthracycline and a cardioprotective agent to the subject, wherein the cardioprotective agent is selected from the group consisting of: SERPINA12; CPVL; PIGR; IQCF3; SESQ2; CSPG2; SHC2; and CFC1 polypeptides or a polynucleotide encoding the polypeptide.

23. A method of treating cardiotoxicity associated with anthracycline treatment in a subject having a neoplasia, the method comprising administering a polypeptide selected from the group consisting of: SERPINA12; CPVL; PIGR; IQCF3; SESQ2; CSPG2; SHC2; and CFC1.

24. The method of any one of claims 13-23, wherein the neoplasia is a sarcoma, breast cancer, stomach cancer, uterine cancer, ovarian cancer, bladder cancer, and / or lung cancer.

25. The method of any one of claims 13-23, wherein the neoplasia is a hematologic malignancy.

26. The method of claim 25, wherein the hematologic malignancy is a leukemia, multiple myeloma (MM), non-Hodgkin lymphoma (NHL), or Hodgkin lymphoma (HL).

27. The method of claim 25, wherein the hematologic malignancy is Diffuse Large B-Cell Lymphoma, Hodgkin’s Lymphoma, Follicular Lymphoma, Acute Myeloid Lymphoma, NonHodgkin’s Lymphoma, T-Cell, Histiocyte Rich Large B Cell Lymphoma, or Aggressive B-Cell Lymphoma.

28. The method of any one of claims 13-23, wherein s a biological sample from the subject is characterized for a polypeptide biomarker selected from the group consisting of or a polynucleotide encoding the polypeptide biomarker.

29. A method of treating a selected subject at risk for heart failure, comprising administering a heart failure treatment, wherein the subject is selected by characterizing a biological sample of the subject as having an alteration in the level of a polypeptide or polynucleotide polypeptidebiomarker relative to a reference, wherein the polypeptide biomarker is selected from the group consisting of carboxypeptidase vitellogenic like (CPVL), polymeric immunoglobulin receptor (PIGR), IQ motif containing F3 (IQCF3), Sesquipedalian-2 (SESQ2), Versican core protein (CSPG2); SHC-transforming protein 2 (SHC2), Cryptic protein (CFC1), Tyrosine-protein phosphatase non-receptor type 2 (TCPTP), Fibroblast growth factor 16 (FGF-16), Aurora kinase B (AURKB), serpin family A member 12 (SERPINA12), Fibroblast growth factor 18 (FGF-18), Carbohydrate sulfotransferase 3 (CHST3), Nectin-1 isoform gamma cytoplasmic domain, Zymogen granule membrane protein 16 (ZG16), Ephrin type-A receptor 10 (EPHAA), Alkaline phosphatase placental type, Serine / threonine protein kinase 4 (STK4), Distintegrin and metalloproteinase domain- containing protein 12 (ADAMI 2), Lupus La protein HTH La-type RNA binding domain, LETM1 domain containing protein (HCCR1), Cytochrome C oxidase subunit 6C (COX6C), Plexin domain containing protein 1 (PXDC1), and Carbonic anhydrase 7.

30. The method of claim 29, wherein the polypeptide biomarkers are CPVL and / or PIGR.

31. The method of claim 29 or claim 30, wherein the level of the polypeptide biomarker is increased relative to a reference.

32. The method of claim 29, wherein the subject is administered a heart failure treatment selected from a beta-blocker, angiotensin-converting enzyme (ACE) inhibitor, angiotensin receptor blocker (ARB), Sodium-Glucose Cotransporter 2 (SGLT2) inhibitor, and / or a mineralicorticoid receptor antagonist.

33. The method of any one of claims 12-32, wherein the anthracycline is selected from doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin, ditrisarubicin, and mitoxantrone, or a pharmaceutically acceptable salt thereof.

34. The method of any one of claims 12-32, wherein the biological sample is a blood sample, serum, or plasma.

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