Biomarker Compositions and Methods of Use Thereof

Abstract

The present disclosure provides methods and kits for identifying and treating individuals at risk of or suffering from amyloid transthyretin cardiomyopathy.Generally, detection or measurement of one or more biomarkers, such as TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or combinations thereof, assists in identifying amyloid transthyretin cardiomyopathy.The present disclosure also provides methods for selecting patients for treatment of amyloid transthyretin cardiomyopathy, such as with a transthyretin stabilizer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119 to or the benefit of U.S. Provisional Application No. 63 / 241,518, filed September 7, 2021, the entire contents of which are incorporated herein by reference.

[0002] Cross-reference to sequence listing This application contains a sequence listing in computer readable form entitled "AttrSEQLST.xml", created on September 7, 2022, which is 12KB in size, and which is incorporated herein by reference.

[0003] The present disclosure relates generally to the fields of molecular biology and cardiac health. [Background technology]

[0004] Amyloid transthyretin cardiomyopathy (TTR-CM) is a rare condition caused by misfolding of the transthyretin protein (TTR), which leads to the deposition of amyloid fibrils in cardiac tissue and ultimately to heart failure. Therefore, early treatment of TTR-CM is important to improve the prognostic impact in affected individuals. However, screening for wild-type transthyretin amyloidosis (ATTRwt) involves multiple costly and invasive procedures that are not particularly specific for TTR-CM (including TTR-CM caused by wild-type ATTR amyloidosis), which can lead to delayed treatment and problems. Furthermore, there is no non-invasive in vitro screening test to detect and / or identify amyloid transthyretin cardiomyopathy (TTR-CM) caused by wild-type ATTR amyloidosis (ATTRwt). Summary of the Invention [Problem to be solved by the invention]

[0005] The difficulty of identifying TTR-CM caused by ATTRwt by current methods may be one of the reasons why this disease is generally underdiagnosed.Therefore, there is a need in the art for compositions, kits and methods for detecting TTR-CM, particularly the TTR-CM caused by wild-type ATTR amyloidosis, with acceptable specificity.In addition, there is a continuous need for compositions, assays, devices and methods for testing, identifying or staging the individual or group of individuals with misfolded TTR protein. [Means for solving the problem]

[0006] The present disclosure provides compositions, kits and methods for detecting and / or identifying TTR-CM, particularly TTR-CM caused by wild-type ATTR amyloidosis. Additionally, the present disclosure provides methods for classifying TTR-CM patients (including TTR-CM caused by wild-type ATTR amyloidosis) in comparison to normal individuals or individuals experiencing other cardiac conditions. Additionally, the present disclosure is the first to recognize that certain biomarkers (e.g., ATTR biomarkers, such as troponin I (TnI), pyruvate kinase muscle isoform 1 (PKM1), pyruvate kinase muscle isoform 2 (PKM2), N-terminal-prohormone B-type natriuretic peptide (NT-proBNP), retinol binding protein 4 (RBP4), decorin (DCN), tissue inhibitor of metalloproteinase 2 (TIMP2), SPARC-related modular calcium binding 2 (SMOC-2), neurofilament light chain (NfL), and combinations thereof) can aid in detecting and / or diagnosing TTR-CM and / or aid in classifying patients with TTR-CM. The present disclosure further recognizes that these biomarkers (e.g., ATTR biomarkers, e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or combinations thereof) in the compositions, kits, and methods may be useful in classifying, detecting, and / or diagnosing TTR-CM (including TTR-CM caused by wild-type ATTR amyloidosis) without the need to perform invasive or costly testing. This represents a significant advancement in patient care, as TTR-CM caused by ATTRwt can be detected and identified by methods that are more comfortable for the patient, cause less harm to the patient, and / or reduce the amount of time the patient requires to recover following the detection and / or diagnostic methods.

[0007] Further, the present disclosure provides that certain biomarkers (e.g., ATTR biomarkers, such as TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or combinations thereof) are useful for detecting TTR-CM with improved sensitivity. The increased sensitivity achieved using the ATTR biomarkers described herein reduces the number of false negatives obtained when detecting and / or identifying TTR-CM. The reduction in false negatives, in turn, helps ensure that more TTR-CM patients receive earlier treatment that is important for reducing the signs, symptoms, and conditions associated with TTR-CM, as well as promoting long-term survival of TTR-CM patients.

[0008] Among other things, the disclosure provides methods that include detecting the levels of each of two or more transthyretin amyloidosis (ATTR) biomarkers in a sample. In some embodiments, the sample is obtained from a subject.

[0009] The present disclosure also provides a method comprising: (a) detecting a level of each of two or more ATTR biomarkers in a sample obtained from a subject to obtain an ATTR biomarker profile; and (b) computing an ATTR biomarker score using the ATTR biomarker profile.

[0010] In some embodiments of the methods provided herein, the two or more ATTR biomarkers comprise (i) troponin I (TnI), (ii) pyruvate kinase muscle isoform 1 (PKM1), (iii) pyruvate kinase muscle isoform 2 (PKM2), (iv) N-terminal-prohormone B-type natriuretic peptide (NT-proBNP), (v) retinol binding protein 4 (RBP4), (vi) tissue inhibitor of metalloproteinase 2 (TIMP2), (vii) neurofilament light chain (NfL), or (viii) a combination thereof.

[0011] In some embodiments, the two or more ATTR biomarkers include TnI and PKM1. In some embodiments, the two or more ATTR biomarkers include TnI and PKM2. In some embodiments, the two or more ATTR biomarkers include TnI, PKM1 and PKM2.

[0012] In some embodiments, the two or more ATTR biomarkers include TnI, PKM2, NT-proBNP, and RBP4. In some embodiments, the two or more ATTR biomarkers include TnI, PKM2, and NT-proBNP. In some embodiments, the two or more ATTR biomarkers include TnI, PKM2, and RBP4. In some embodiments, the two or more ATTR biomarkers include TnI, PKM1, and NT-proBNP. In some embodiments, the two or more ATTR biomarkers include TnI, PKM1, and RBP4. In some embodiments, the two or more ATTR biomarkers include TnI, PKM1, PKM2, NT-proBNP, and RBP4. In some embodiments, the two or more ATTR biomarkers include TnI, PKM1, PKM2, and NT-proBNP. In some embodiments, the two or more ATTR biomarkers include TnI, PKM1, PKM2, and RBP4. In some embodiments, the two or more ATTR biomarkers include NT-proBNP, RBP4, and TnI. In some embodiments, the two or more ATTR biomarkers include RBP4, SMOC-2, and TnI. In some embodiments, the two or more ATTR biomarkers include DCN, NT-proBNP, and TnI. In some embodiments, the two or more ATTR biomarkers include DCN, RBP4, and TnI. In some embodiments, the two or more ATTR biomarkers include NT-proBNP, SMOC-2, and TnI. In some embodiments, the two or more ATTR biomarkers include NT-proBNP, TIMP2, and TnI. In some embodiments, the two or more ATTR biomarkers include NT-proBNP and TnI. In some embodiments, the two or more ATTR biomarkers include DCN and TnI. In some embodiments, the two or more ATTR biomarkers include DCN, TIMP2, and TnI. In some embodiments, the two or more ATTR biomarkers include RBP4 and TnI. In some embodiments, the two or more ATTR biomarkers include RBP4, TIMP2, and TnI.In some embodiments, the two or more ATTR biomarkers include DCN, SMOC-2, and TnI. In some embodiments, the two or more ATTR biomarkers include TIMP2 and TnI. In some embodiments, the two or more ATTR biomarkers include SMOC-2, TIMP2, and TnI.

[0013] In some embodiments, the two or more ATTR biomarkers do not include PKM1. In some embodiments, the two or more ATTR biomarkers do not include PKM2. In some embodiments, the two or more ATTR biomarkers do not include PKM1 or PKM2.

[0014] In some embodiments, the two or more ATTR biomarkers do not include SMOC-2. In some embodiments, the two or more ATTR biomarkers do not include DCN. In some embodiments, the two or more ATTR biomarkers do not include SMOC-2 or DCN.

[0015] In some embodiments, using the ATTR biomarker profile to compute the ATTR biomarker score comprises applying an algorithm to the ATTR biomarker profile to compute the ATTR biomarker score. In some embodiments, the algorithm is or is derived from a decision tree method, a neural boosting method, a bootstrap forest method, a boosted tree method, a K-nearest neighbor method, a generalized regression forward selection methodology, a generalized regression pruned forward selection methodology, a fit stepwise methodology, a generalized regression lasso method, a generalized regression elastic net method, a generalized regression ridge method, a nominal logistic method, a support vector machine method, a discriminant method, a naive Bayes method, or a combination thereof. In some embodiments, the algorithm is or is derived from a decision tree method, a neural boosting method, a bootstrap forest method, a boosted tree method, a generalized regression lasso method, a generalized regression elastic net method, a generalized regression ridge method, a nominal logistic method, a support vector machine method, a discriminant method, or a combination thereof. In some embodiments, the algorithm is or is derived from a decision tree method, a neural boosting method, a bootstrap forest method, a boosted tree method, a support vector machine method, or a combination thereof.

[0016] In some embodiments, the methods described herein include using the score of the ATTR biomarkers to determine whether the subject from whom the sample was obtained is at risk for or suffering from transthyretin amyloid cardiomyopathy (TTR-CM). In some embodiments, the methods described herein include using the score of the ATTR biomarkers to diagnose the subject with TTR-CM. In some embodiments, the methods described herein include using the score of the ATTR biomarkers to determine whether the subject from whom the sample was obtained is selected for one or more cardiomyopathy trials. In some embodiments, the methods described herein include using the score of the ATTR biomarkers to determine whether the subject from whom the sample was obtained is selected to receive one or more doses of a TTR stabilizer.

[0017] In some embodiments, the subject is a human subject.

[0018] In some embodiments, the sample comprises blood, serum, plasma, or cardiac tissue.

[0019] The present disclosure also provides a non-transitory computer readable medium. In some embodiments, the non-transitory computer readable medium includes executable instructions that, when executed, cause a processor to perform operations, including the methods described herein.

[0020] Furthermore, the present disclosure provides a composition.In some embodiments, the composition comprises one or more ATTR biomarkers.In some embodiments, the one or more ATTR biomarkers comprise TnI, PKM1, PKM2, NT-proBNP, RBP4, TIMP2, NfL or combinations thereof.

[0021] In some embodiments, the composition comprises one or more anti-ATTR biomarker drugs. In some embodiments, the one or more anti-ATTR biomarker drugs comprise an anti-TnI drug, an anti-PKM1 drug, an anti-PKM2 drug, an anti-NT-proBNP drug, an anti-RBP4 drug, an anti-TIMP2 drug, an anti-NfL drug, or a combination thereof.

[0022] The present disclosure further provides a kit.In some embodiments, the kit comprises one or more ATTR biomarkers.In some embodiments, the one or more ATTR biomarkers comprise TnI, PKM1, PKM2, NT-proBNP, RBP4, TIMP2, NfL or combinations thereof.

[0023] In some embodiments, the kit comprises one or more anti-ATTR biomarker drugs. In some embodiments, the one or more anti-ATTR biomarker drugs comprise an anti-TnI drug, an anti-PKM1 drug, an anti-PKM2 drug, an anti-NT-proBNP drug, an anti-RBP4 drug, an anti-TIMP2 drug, an anti-NfL drug, or a combination thereof.

[0024] In some embodiments, the kit comprises instructions for use.

[0025] In some embodiments, the kit comprises one or more anti-ATTR biomarker drugs, wherein the one or more anti-ATTR biomarkers comprise one or more antibody drugs. In some embodiments, one or more of the antibody drugs are labeled with a detectable moiety.

[0026] In some embodiments, the kit comprises one or more control samples. In some embodiments, the control samples comprise one or more ATTR biomarker standards.

[0027] The present disclosure provides the use of the kit described herein. In some embodiments, the kit can be used in an in vitro diagnostic assay for diagnosing TTR-CM in a subject. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 illustrates a patient cohort used in an exemplary method described herein. [Diagram 2]FIG. 1 shows a summary of data obtained from an exemplary method described herein. [Diagram 3] Figure 3A includes graphs showing the individual sensitivity and specificity performances as determined with TIMP2 and Figure 3B includes graphs showing the individual sensitivity and specificity performances as determined with TnI. [Figure 4] Figure 4A includes graphs showing the individual sensitivity and specificity performances as determined by PKM and Figure 4B includes graphs showing the individual sensitivity and specificity performances as determined by RBP4. [Diagram 5] Figure 5A includes graphs showing the performance of individual sensitivities and specificities as determined by RBP and Figure 5B includes graphs showing the performance of individual sensitivities and specificities as determined by DCN. [Figure 6] Figure 6A includes graphs showing the performance of each sensitivity and specificity determined with NT-proBNP and Figure 6B includes graphs showing the performance of each sensitivity and specificity determined with SMOC-2. [Figure 7] FIG. 1 includes bar graphs showing the effect of representative biomarkers on each NYHA class. [Figure 8-1] FIG. 1 includes a table showing the results of screening with exemplary biomarkers described herein, including an assessment of the regression fits used. [Figure 8-2] Continued from Figure 8-1. [Figure 9] FIG. 1 includes tables outlining the data obtained when a selected subset of biomarkers was used in various predictive models. [Figure 10] Figure 10 shows the optimization of detection cut-off values ​​for assays measuring PKM, TIMP2, LIMS-1, C3 and A11. Figure 10A depicts ROC plot data for optimization of detection cut-off values. Figure 10B depicts exemplary values ​​of sensitivity, specificity, accuracy and detection cut-off values ​​for the listed biomarkers. [Figure 11] FIG. 11 depicts an exemplary block diagram of a computer system 1100. [Figure 12] 12 depicts an exemplary flowchart of a method 1200. [Figure 13] 13 depicts an exemplary flowchart of a method 1300. [Figure 14-1] FIG. 1 includes a table showing the signal to noise ratios of selected markers. [Figure 14-2] Continued from Figure 14-1. [Figure 14-3] Continued from Figure 14-2. [Figure 14-4] Continued from Figure 14-3. [Figure 14-5] Continued from Figure 14-4. [Figure 14-6] Continued from Figure 14-5. [Figure 14-7] Continued from Figure 14-6. [Figure 14-8] Continued from Figure 14-7. [Figure 14-9] Continued from Figure 14-8. [Figure 14-10] Continued from Figure 14-9. [Figure 14-11] Continued from Figure 14-10. [Figure 14-12] Continued from Figure 14-11. [Figure 14-13] Continued from Figure 14-12. [Figure 14-14] Continued from Figure 14-13. [Figure 14-15] Continued from Figure 14-14. [Figure 14-16] Continued from Figure 14-15. [Figure 14-17] Continued from Figure 14-16. [Figure 14-18] Continued from Figure 14-17. [Figure 14-19] Continued from Figure 14-18. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] definition Antibody Drug: As used herein, the term "antibody drug" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Such polypeptides can be naturally generated (e.g., generated by an organism that responds to an antigen) or can be generated by recombinant engineering, chemical synthesis or other artificial systems or methodologies. Exemplary antibody drugs include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, conjugated antibodies (e.g., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), Small Modular ImmunoPharmaceuticals ("SMIPs™"), single chain antibodies, cameloid antibodies, and antibody fragments. As used herein, the term "antibody drug" includes intact monoclonal antibodies, polyclonal antibodies, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or fragments thereof)), multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. An antibody drug can have an antibody constant region sequence characteristic of a mouse, rabbit, primate or human antibody. In some embodiments, the term includes staple peptides. In some embodiments, the term includes one or more antibody-like binding peptidomimetics. In some embodiments, the term includes one or more antibody-like binding scaffold proteins. In some embodiments, the term includes monobodies or adnectins.In many embodiments, an antibody drug is or comprises a polypeptide whose amino acid sequence comprises one or more structural elements recognized by those of skill in the art as a complementarity determining region (CDR); in some embodiments, an antibody drug is or comprises a polypeptide whose amino acid sequence comprises at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) substantially identical to that found in a reference antibody. In some embodiments, an antibody drug is or comprises a polypeptide whose amino acid sequence comprises a structural element recognized by those of skill in the art as an immunoglobulin variable domain. In some embodiments, an antibody drug is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. In some embodiments, an antibody drug can comprise a covalent modification (e.g., glycan, payload (e.g., detectable moiety, therapeutic moiety, catalytic moiety, etc.), or other pendant group (e.g., attachment of polyethylene glycol, etc.).

[0030] Biomarker: The term "biomarker" or "biological marker" is used herein, consistent with its use in the art, to refer to an entity whose presence, level, or form correlates with a particular biological event or condition of interest and is therefore considered a "marker" for that event or condition. To name just a few examples, in some embodiments, a biomarker may be or include a marker for a particular pathology, or for the likelihood that a particular disease, disorder, or condition will occur, occur, or reoccur. In some embodiments, a biomarker may be or include a marker for a particular disease or treatment outcome or the likelihood thereof. Thus, in some embodiments, a biomarker is predictive of a relevant biological event or condition of interest, in some embodiments, a biomarker is prognostic of the biological event or condition, and in some embodiments, a biomarker is diagnostic of the biological event or condition. In some embodiments, a biomarker is a possible biomarker of a relevant biological event or condition of interest. A biomarker may be an entity of any chemical class. For example, in some embodiments, a biomarker may be or include a nucleic acid, a polypeptide, a small molecule, or a combination thereof. In some embodiments, the biomarker is a cell surface marker. In some embodiments, the biomarker is intracellular. In some embodiments, the biomarker is found in a particular tissue (e.g., cardiac tissue). In some embodiments, the biomarker is found outside the cell (e.g., secreted or otherwise produced or present outside the cell, e.g., in bodily fluids, e.g., blood, urine, tears, saliva, cerebrospinal fluid, etc.).

[0031] As described herein, in some embodiments, the biomarker is an ATTR biomarker. As used herein, "ATTR biomarker" refers to a biological marker for ATTR amyloidosis or TTR-CM. In some embodiments, one or more ATTR biomarkers include TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. For the avoidance of doubt, the ATTR biomarker includes gene products associated with the specific biomarkers listed. For example, depending on the context, "TnI" refers to the nucleotides encoding TnI or a characteristic fragment thereof, as well as the TnI protein or a characteristic fragment thereof.

[0032] Characteristic fragment: The term "characteristic fragment" refers to a fragment of a biomarker (e.g., the ATTR biomarker) that is sufficient to identify the biomarker from which the fragment is derived. For example, in some embodiments, a "characteristic fragment" of a biomarker is one that comprises an amino acid sequence or collection of amino acid sequences that together allow for distinguishing the biomarker from which the fragment is derived from other possible biomarkers, proteins or polypeptides. In some embodiments, a characteristic fragment comprises at least 10, at least 20, at least 30, at least 40 or at least 50 amino acids.

[0033] Gene product or expression product: As used herein, the term "gene product" generally refers to the RNA transcribed from a gene (before and / or after processing) or the polypeptide encoded by the RNA transcribed from a gene (before and / or after modification).

[0034] Hybridization: The term "hybridization" refers to the physical property of a single-stranded nucleic acid molecule (e.g., DNA or RNA) to anneal to a complementary nucleic acid molecule. Hybridization can typically be assessed in a variety of contexts, including when interacting nucleic acid molecules are studied independently or in the context of a more complex system (e.g., while covalently or otherwise bound to a carrier entity and / or in a biological system or cell). In some embodiments, hybridization can be detected by a hybridization technique, such as a technique selected from the group consisting of in situ hybridization (ISH), microarray, Northern blot, and Southern blot. In some embodiments, hybridization refers to 100% annealing between a single-stranded nucleic acid molecule and a complementary nucleic acid molecule. In some embodiments, annealing is less than 100% (e.g., at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70% of the single-stranded nucleic acid molecule anneals to a complementary nucleic acid molecule). Hybridization techniques and methods for evaluating hybridization are well known in the art.See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Plainview, NY. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions so that sequences with at least the desired level of complementarity will stably hybridize, but those with lower complementarity will not stably hybridize.See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al., 1994, Current Protocols in Molecular Biology.John Wiley & Sons, Secaucus, NJ.

[0035] Detection Agent: As used herein, the term "detection agent" refers to any element, molecule, functional group, compound, fragment, or moiety that is capable of being detected. In some embodiments, a detection agent is provided or utilized alone. In some embodiments, a detection agent is provided and / or utilized in conjunction with (e.g., bound to) another agent. Examples of detection agents include, but are not limited to, various ligands, radionuclides (e.g., 3 H, 14 C. 18 F, 19 F, 32 P, 35 S, 135 I, 125 I, 123 I, 64 Cu, 187 Re, 111 In, 90 Y, 99m Tc, 177 Lu, 89 Zr, etc.), fluorescent dyes, chemiluminescent agents (e.g., acridinium esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (e.g., dyes, colloidal gold, etc.), biotin, digoxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[0036] Diagnostic test: As used herein, a "diagnostic test" is a step or series of steps that is or has been performed to obtain information useful for determining whether a patient has a disease, disorder or condition, and / or for classifying a disease, disorder or condition into a phenotypic category, or into any category that has significance for the prognosis of the disease, disorder or condition, or the likely response to treatment of the disease, disorder or condition (either in general or any specific treatment). Similarly, "diagnosis" refers to providing any type of diagnostic information, including but not limited to, whether a subject has or is likely to develop a disease, disorder or condition, the status, staging, or characteristics of the disease, disorder or condition manifested in the subject, information regarding the nature or classification of the tumor, information regarding the prognosis, and / or information useful for selecting an appropriate treatment or further diagnostic tests. Selection of treatment can include selection of a particular therapeutic or other treatment, surgery, radiation, etc., selection of whether to withhold or provide therapy, selection of dosing regimen (e.g., frequency or level of one or more doses of a particular therapeutic or combination of therapeutics), etc. Selection of further diagnostic tests can include more specific tests for a given disease, disorder, or condition.

[0037] Sample: As used herein, the term "sample" refers to a biological sample obtained from or derived from a human subject, as described herein. In some embodiments, the biological sample comprises a biological tissue or fluid. In some embodiments, the biological sample can comprise blood; blood cells; tissue or fine needle biopsy samples; cell-containing bodily fluids; free-floating nucleic acids; cerebrospinal fluid; lymphatic fluid; tissue biopsy specimens; surgical specimens; other bodily fluids, secretions and / or excretions; and / or cells derived therefrom. In some embodiments, the biological sample comprises cells obtained from an individual, e.g., from a human or animal subject. In some embodiments, the cells obtained are or comprise cells from the individual from whom the sample is obtained. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, and collection of bodily fluids (e.g., blood). In some embodiments, the sample is cardiac tissue obtained from a subject. In some embodiments, as is clear from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample), e.g., by filtering using a semi-permeable membrane. As another example of sample processing, the sample may be a plasma sample, which is treated with an anticoagulant selected from the group consisting of EDTA, heparin, and citrate. As another example of sample processing, the sample may be treated to isolate one or more proteins (e.g., by capturing the proteins with one or more antibodies). A "processed sample" may include, for example, nucleic acids or polypeptides extracted from the sample or obtained by subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components.

[0038] Subject: As used herein, the term "subject" refers to an organism, e.g., a mammal (e.g., a human). In some embodiments, the human subject is an adult, adolescent, or pediatric subject. In some embodiments, the subject is at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, or at least 80 years old. In some embodiments, the subject suffers from a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein. In some embodiments, the subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is prone to a disease, disorder, or condition and / or exhibits an increased risk of developing a disease, disorder, or condition (compared to the average risk observed in a reference subject or population). In some embodiments, the subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, the subject does not exhibit a particular symptom (e.g., clinical symptoms of a disease) or characteristic of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, a subject is an individual to whom and / or who has been administered a diagnosis and / or therapy.

[0039] Threshold: As used herein, the term "threshold" refers to a value used as a reference to obtain information and / or classify the results of a measurement, e.g., the results of a measurement obtained in an assay. The threshold can be determined based on one or more control samples. The threshold can be determined prior to, at the same time as, or after the measurement of interest is performed. In some embodiments, the threshold can be a range of values. In some embodiments, the threshold can be a value (or range of values) reported in the relevant field (e.g., values ​​found in standard tables).

[0040] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Amyloid transthyretin cardiomyopathy Transthyretin (TTR) is a transport protein found in serum and cerebrospinal fluid that transports the thyroid hormone thyroxine (T4) and retinol-binding protein. The liver secretes TTR into the blood, and the choroid plexus secretes TTR into the cerebrospinal fluid. TTR is produced as a homotetrameric complex. However, TTR can undergo conformational transformation and aggregate into abnormal amyloid forms that lead to pathological conditions.

[0041] ATTR amyloidosis is characterized by the deposition of amyloid fibrils derived from transthyretin (TTR) protein in various organs and tissues. For example, misfolding of TTR protein can cause the deposition of amyloid fibrils in cardiac tissue, leading to cardiomyopathy (referred to herein as "amyloid transthyretin cardiomyopathy", or "TTR-CM"). Clinical symptoms of TTR-CM include increased ventricular wall thickness and heart failure.

[0042] There are three types of ATTR amyloidosis: (1) familial amyloid polyneuropathy (FAP), (2) familial amyloid cardiomyopathy (FAC), and (3) senile systemic amyloidosis, also called wild-type ATTR amyloidosis (ATTRwt). Familial amyloid polyneuropathy (FAP) affects the nervous system in addition to the heart and sometimes the kidneys and eyes. Symptoms of FAP can include peripheral neuropathy, autonomic neuropathy, and heart failure. Familial amyloid cardiomyopathy (FAC) affects the heart and is further manifested with carpal tunnel syndrome. FAP and FAC are inherited conditions caused by mutations in the TTR gene that lead to the production of abnormal ("variant") TTR. Over 100 different mutations have been observed in the TTR gene, many of which lead to the production of variant TTR that can misfold into amyloid fibrils and cause the aggregation of amyloid deposits in tissues. Most affected individuals are heterozygotes; therefore, both mutant and wild-type TTR may be included in the aggregate. As used herein, the hereditary cardiomyopathy caused by mutant or variant forms of TTR is referred to as familial amyloid cardiomyopathy (abbreviated ATTRm). Wild-type ATTR amyloidosis (ATTRwt) is a slowly progressive non-hereditary (sporadic) disease. Individuals with ATTRwt do not have mutations in the TTR gene, and amyloid fibrils are composed of wild-type TTR. Symptoms of ATTRwt include heart failure and, in some individuals, carpal tunnel syndrome. ATTRwt occurs more frequently in elderly subjects over 65 years of age.

[0043] Although the exact prevalence is unknown, ATTRm is roughly estimated at 0.4 cases / million people / year in the United States, with an incidence rate at least two times higher for ATTRwt. However, autopsy studies of patients over 80 years of age have shown a prevalence of approximately 25% for ATTRwt, suggesting that the disease is largely underdiagnosed, and that the actual prevalence is much higher, especially in the elderly population. The challenge of testing large numbers of individuals with costly and invasive tests and procedures is a possible contributing factor to the significant underdiagnosis of TTR-CM.

[0044] Current diagnostic methods for amyloid transthyretin cardiomyopathy TTR-CM is currently diagnosed using a series of different tests starting with an echocardiogram (Gertz, MA, et al., JACC 66:2452-2466, 2015). This technique is used to confirm general cardiac function and to look for structural abnormalities (Ashley, EA and Niebauer, J., Cardiology Explained, London: Remedica, Chapter 4, 2004), and as a screen for cardiac amyloidosis, evidenced by ventricular thickening and hypertrophy. The test cannot distinguish between hypertrophic and hypertensive cardiomyopathy, but has shown relatively good specificity (e.g., 82%) in distinguishing cardiac amyloidosis from cardiac hypertrophy in one study (Ashley 2004). However, since there is more than one form of cardiac amyloidosis (e.g., light chain amyloidosis (AL)), the test is not specific for TTR-CM.

[0045] Patients who test positive on echocardiogram are then examined by cardiac magnetic resonance imaging (CMR) (Gertz, MA, et al., JACC 66:2452-2466, 2015; Krishnamurthy, R., et al., Current Cardiology Reviews, 9:185-190, 2013; Doltra, A., et al., Curr Cardiol Rev. 9(3):185-90, 2013). This technique uses the contrast agent gadolinium, which can focus in areas of cardiac cell damage (e.g., myocardial infarction) or areas with increased extracellular space due to scarring or amyloid deposits (Krishnamurthy, R., et al., Current Cardiology Reviews, 9:185-190, 2013). This technique can distinguish between hypertensive and hypertrophic cardiomyopathy better than echocardiograms and in one study showed greater specificity for detecting ATTR amyloidosis than AL (Ashley 2004). However, this test is not by itself a definitive test for the diagnosis of TTR-CM.

[0046] Another imaging method for the detection of cardiac amyloidosis is 99m Scintigraphy uses radioisotope conjugates such as Tc-pyrophosphate (Bokhari et al., Circ Cardiovasc Imaging. 6(2):195-201, 2013). It is performed using single photon emission computed tomography (SPECT) to obtain 3D images, and although the radioactive tracer is not specific for cardiac tissue, it is useful for detecting areas of poor blood flow such as occurs in diseased cardiac tissue. This technique has been shown in one study to distinguish ATTR amyloidosis from AL amyloidosis with high specificity and sensitivity (100% and 97%, respectively).

[0047] Currently, the most definitive test for the diagnosis of TTR-CM, typically performed after a positive score is obtained using the tests described above, is a cardiac biopsy followed by immunochemical staining. Polyclonal antibodies against kappa or light chain amyloid deposits in cardiac tissue are used to detect AL, while polyclonal antibodies against transthyretin deposits are used to detect TTR-CM (Crotty, TB et al., Cardiovacular Pathology 4:39-42, 1995).

[0048] Finally, antibodies against transthyretin (TTR) have been previously disclosed and the application of such antibodies in the diagnosis of amyloid diseases such as ATTRwt has been described (WO2014 / 124334A2 and WO2016 / 120811). A disadvantage to using such anti-TTR antibodies for the diagnosis of cardiomyopathies such as those caused by ATTRwt is that TTR may not be recognized in a misfolded or aggregated context in some patients, resulting in false negative test results and underdiagnosis. For example, both WO2014 / 124334A2 and WO2016 / 120811 rely on the exposure of epitopes within specific amino acid residues of TTR; however, such epitopes may not be readily accessible in all ATTR amyloidosis patients due to non-native forms of TTR. Wild-type TTR, mutant TTR or mixed TTR tetramers may dissociate, misfold, aggregate and / or form fibrils in ATTR amyloidosis disease. Such different forms may not be easily detected with anti-TTR antibodies. In addition, the gene encoding TTR has been reported to have many different mutations associated with ATTR amyloidosis. Therefore, anti-TTR antibodies may not be able to recognize the disease in many patients.

[0049] ATTR biomarkers The embodiments described herein provide several advantages over the conventional techniques discussed herein. For example, the techniques disclosed herein are non-invasive, require minimal patient discomfort, are performed quickly, and are relatively cost-effective. Thus, the techniques described herein provide advantages over these conventional techniques, including but not limited to providing: a non-invasive in vitro diagnostic (IVD) test for TTR-CM caused by ATTRwt, one or more specific in vitro biomarkers suitable for use in IVD testing, an alternative to single-marker IVD testing, including one or more markers that effectively rule in or rule out candidates for more expensive and invasive procedures in diagnosing disease.

[0050] The present disclosure particularly relates to biomarkers for ATTR amyloidosis and TTR-CM. Such biomarkers are referred to herein as "ATTR biomarkers". ATTR biomarkers can include, for example, TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, characteristic fragments thereof, and / or variants thereof.

[0051] As provided herein, ATTR biomarkers include gene products associated with the specific biomarkers listed. For example, ATTR biomarkers can include, for example, proteins or nucleotides (e.g., RNA, e.g., mRNA). ATTR biomarkers also encompass full-length proteins and fragments (e.g., characteristic fragments) of ATTR biomarkers. For example, in some embodiments, ATTR biomarkers can include proteins listed in Table 1. In some embodiments, ATTR biomarkers include fragments having an amino acid sequence identical to a contiguous span of at least 10 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or at least 100 amino acids of the amino acid sequence provided in Table 1. In some embodiments, ATTR biomarkers include fragments having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence provided in Table 1. Variants or alternative forms of the biomarkers include, for example, polypeptides encoded by any splice variants of the transcripts encoding the disclosed biomarkers.

[0052] Biomarkers contemplated herein also include truncated forms or polypeptide fragments of any of the proteins described herein. Truncated forms or polypeptide fragments of proteins can include N-terminally removed or truncated forms and C-terminally removed or truncated forms. Truncated forms or fragments of proteins can include fragments generated by any mechanism, including but not limited to selective translation, exo- and / or endo-proteolysis, and / or degradation, for example, by physical, chemical and / or enzymatic proteolysis. Without limitation, biomarkers can also include truncated forms or fragments of proteins, polypeptides or peptides, which can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 8%, or at least 99% of the amino acid sequence of an ATTR biomarker protein.

[0053] In some cases, the fragments are N-terminally and / or C-terminally truncated by 1-20 amino acids, e.g., by only 1-15 amino acids, 1-10 amino acids, or 1-5 amino acids, compared to the corresponding mature full-length ATTR biomarker protein.

[0054] Any ATTR biomarker proteins of the present disclosure, such as peptides, polypeptides or proteins and fragments thereof, can also include modified forms of said markers, peptides, polypeptides or proteins and fragments, such as having post-expression modifications, including, but not limited to, modifications such as phosphorylation, glycosylation, lipidation, methylation, selenocystine modification, cysteinylation, sulfonation, glutathionylation, acetylation, and / or oxidation of methionine to methionine sulfoxide or methionine sulfone.

[0055] In some embodiments, the ATTR biomarker has different isoforms. Although only one or more isoforms may be disclosed herein, all isoforms of the ATTR biomarker are contemplated for use in the disclosed technology.

[0056] In some embodiments, the ATTR biomarker can be nucleotide. In some embodiments, the nucleotide can be RNA or DNA. In some cases, the corresponding RNA or DNA can show better discrimination in diagnosis compared with full-length protein.

[0057] Exemplary ATTR biomarkers for use with the technology provided herein are briefly described below.

[0058] Troponin I (TnI) Troponins are a group of proteins found in skeletal and cardiac muscle fibers. One function of troponins is to regulate muscle contraction. Three types of troponin proteins are known: troponin C, troponin I, and troponin T. Together, the three types of troponins form a complex. Within the complex, troponin C binds calcium ions. This binding causes a conformational change in troponin I, thereby causing contraction. Troponin I binds to actin in thin myofilaments, holding the actin-tropomyosin complex in place. Troponin T anchors the troponin complex to tropomyosin, a muscle fiber structure.

[0059] Previous analysis indicates that there is little or no difference in troponin C between skeletal and cardiac muscles, while the forms of troponin I and troponin T are understood to be different between skeletal and cardiac muscles. Normally, troponin is present in the blood in very small to undetectable amounts. However, when there is damage to cardiac muscle cells, troponin is released into the blood. Higher concentrations of troponin I in the blood generally correlate with greater damage to cardiac tissue.

[0060] In some embodiments of the present disclosure, TnI is an ATTR biomarker. In some embodiments, detection of TnI, a characteristic fragment of TnI, and / or a variant of TnI is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, TnI is detected in a sample using an anti-TnI drug (e.g., an anti-TnI antibody drug, a probe, etc.). In some embodiments, detection of nucleotides encoding TnI, nucleotides encoding characteristic fragments of TnI, and / or nucleotides encoding variants of TnI is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, nucleotides encoding TnI are detected in a sample using an anti-TnI nucleotide sequence drug (e.g., an anti-TnI nucleotide sequence antibody drug, a probe, a complementary nucleic acid, etc.).

[0061] Pyruvate kinase muscle isoform 1 and isoform 2 (PKM1 and PKM2) Pyruvate kinase is an enzyme that catalyzes the final step of glycolysis by catalyzing the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP) to yield pyruvate and ATP. In vertebrates, there are four tissue-specific isozymes of pyruvate kinase: L (liver), R (erythrocytes), muscle isoform 1 (muscle, heart and brain) and muscle isoform 2 (early fetal tissues and most adult tissues). Pyruvate kinase proteins can form dimers and tetramers.

[0062] The PKM gene encodes muscle isoform 1 and muscle isoform 2 isozymes (PKM1 and PKM2). Exons 9 and 10 of the PKM gene contain sequences for muscle isoform 1 and muscle isoform 2 isozymes, respectively. There are at least 14 splice variants of PKM, including one non-coding variant. Among the splice variants of PKM are PKM1 and PKM2, which are produced by differential splicing and differ by 22 amino acids at the carboxy terminus. Because the amino acid sequences of PKM1 and PKM2 share an identical region, certain fragments of PKM1 and PKM2 are characteristic fragments of both PKM1 and PKM2, and certain fragments (e.g., fragments from the 22 amino acids at the carboxy terminus) are characteristic fragments of PKM1 or PKM2. Furthermore, because the amino acid sequences of PKM1 and PKM2 share an identical region, certain anti-PKM1 drugs also detect PKM2, and vice versa.

[0063] In some embodiments of the present disclosure, PKM (e.g., PKM1 and / or PKM2) is an ATTR biomarker. In some embodiments, detection of PKM, a characteristic fragment of PKM, and / or a variant of PKM is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, PKM is detected in a sample using an anti-PKM drug (e.g., an anti-PKM antibody drug, a probe, etc.). In some embodiments, detection of nucleotides encoding PKM, a characteristic fragment of PKM, and / or a variant of PKM is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, nucleotides encoding PKM are detected in a sample using an anti-PKM nucleotide sequence drug (e.g., an anti-PKM nucleotide sequence antibody drug, a probe, a complementary nucleic acid, etc.).

[0064] In some embodiments of the present disclosure, PKM1 is an ATTR biomarker. In some embodiments, detection of PKM1, a characteristic fragment of PKM1, and / or a variant of PKM1 is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, PKM1 is detected in a sample using an anti-PKM1 drug (e.g., an anti-PKM1 antibody drug, a probe, etc.). In some embodiments, detection of nucleotides encoding PKM1, nucleotides encoding characteristic fragments of PKM1, and / or nucleotides encoding variants of PKM1 is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, nucleotides encoding PKM1 are detected in a sample using an anti-PKM1 nucleotide sequence drug (e.g., an anti-PKM1 nucleotide sequence antibody drug, a probe, a complementary nucleic acid, etc.).

[0065] In some embodiments of the present disclosure, PKM2 is an ATTR biomarker. In some embodiments, detection of PKM2, a characteristic fragment of PKM2, and / or a variant of PKM2 is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, PKM2 is detected in a sample using an anti-PKM2 drug (e.g., an anti-PKM2 antibody drug, a probe, etc.). In some embodiments, detection of nucleotides encoding PKM2, a characteristic fragment of PKM2, and / or a variant of PKM2 is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, nucleotides encoding PKM2 are detected in a sample using an anti-PKM2 nucleotide sequence drug (e.g., an anti-PKM2 nucleotide sequence antibody drug, a probe, a complementary nucleic acid, etc.).

[0066] N-terminal prohormone B-type natriuretic peptide (NT-proBNP) B-type natriuretic peptide (BNP) is a hormone produced in the heart. BNP is a small cyclic peptide secreted by the heart to regulate blood pressure and fluid balance. N-terminal (NT)-precursor hormone BNP (NT-proBNP or NT-proBNP) is an inactive hormone precursor released from the same molecule that produces BNP. In particular, BNP is stored in a precursor form (proBNP) in membrane granules of the ventricles from which it is primarily secreted. Once released from the heart in response to ventricular volume expansion or pressure overload, the 76 amino acid N-terminal (NT) fragment (NT-proBNP) is rapidly cleaved by the enzymes corin and furin to release the active 32 amino acid peptide (BNP).

[0067] In some embodiments of the present disclosure, NT-proBNP is an ATTR biomarker. In some embodiments, detection of NT-proBNP, a characteristic fragment of NT-proBNP, and / or a variant of NT-proBNP is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, NT-proBNP is detected in a sample using an anti-NT-proBNP drug (e.g., an anti-NT-proBNP antibody drug, a probe, etc.). In some embodiments, detection of nucleotides encoding NT-proBNP, a characteristic fragment of NT-proBNP, and / or a variant of NT-proBNP is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, nucleotides encoding NT-proBNP are detected in a sample using an anti-NT-proBNP nucleotide sequence drug (e.g., an anti-NT-proBNP nucleotide sequence antibody drug, a probe, a complementary nucleic acid, etc.).

[0068] Retinol-binding protein 4 (RBP4) Retinol-binding protein 4 (RBP4) is a transporter protein for retinol (vitamin A alcohol). RBP4 has a molecular weight of approximately 21 kDa and is encoded by the RBP4 gene in humans. It is primarily, but not exclusively, synthesized in the liver. RBP4 delivers retinol from storage sites in the liver to peripheral tissues. In plasma, the RBP-retinol complex interacts with transthyretin, which prevents its loss by filtration through the kidney glomerulus. Vitamin A deficiency post-translationally blocks the secretion of this binding protein, resulting in incomplete delivery and supply to epithelial cells. Circulating RBP4 has previously been proposed as a way to distinguish between ATTRm and nonamyloid heart failure (Arvanitis, M. et al., JAMA Cardiol., 2017). However, this prior study only looked at ATTRm caused by a specific mutation in RBP4 (a substitution of isoleucine for valine at codon 122 of the TTR gene (V122I)) and did not examine whether RBP4 could be used more generally as a biomarker for TTR-CM, whether assessed alone or together with other possible biomarkers.

[0069] The present disclosure provides the recognition that RBP4 can be used as an ATTR biomarker. Thus, in some embodiments of the present disclosure, RBP4 is an ATTR biomarker. In some embodiments, detection of RBP4, characteristic fragments of RBP4, and / or variants of RBP4 is used in a method for assessing the risk of a subject developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, RBP4 is detected in a sample using an anti-RBP4 drug (e.g., anti-RBP4 antibody drug, probe, etc.). In some embodiments, detection of nucleotides encoding RBP4, characteristic fragments of RBP4, and / or variants of RBP4 is used in a method for assessing the risk of a subject developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, nucleotides encoding RBP4 are detected in a sample using an anti-RBP4 nucleotide sequence drug (e.g., anti-RBP4 nucleotide sequence antibody drug, probe, complementary nucleic acid, etc.).

[0070] Tissue inhibitor of metalloproteinase 2 (TIMP2) Tissue inhibitor of metalloproteinase 2 (TIMP2) is the gene that encodes the TIMP2 protein. The TIMP2 gene is encoded by five exons spanning 83 kb of genomic DNA. The 5 prime end of the TIMP2 gene contains several regulatory elements, including Sp1 binding site, AP2 binding site, AP1 binding site and PEA3 binding site.

[0071] The TIMP2 gene is a member of the TIMP gene family. Proteins encoded by genes in the TIMP gene family inhibit matrix metalloproteinases (MMPs), a group of peptidases involved in the degradation of extracellular matrix. TIMP2 also has the ability to directly suppress endothelial cell proliferation. TIMP2 has been shown to suppress tumor metastasis.

[0072] In some embodiments of the present disclosure, TIMP2 is an ATTR biomarker. In some embodiments, detection of TIMP2, a characteristic fragment of TIMP2, and / or a variant of TIMP2 is used in a method for assessing the risk of a subject developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, TIMP2 is detected in a sample using an anti-TIMP2 drug (e.g., an anti-TIMP2 antibody drug, a probe, etc.). In some embodiments, detection of nucleotides encoding TIMP2, nucleotides encoding characteristic fragments of TIMP2, and / or nucleotides encoding variants of TIMP2 is used in a method for assessing the risk of a subject developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, nucleotides encoding TIMP2 are detected in a sample using an anti-TIMP2 nucleotide sequence drug (e.g., an anti-TIMP2 nucleotide sequence antibody drug, a probe, a complementary nucleic acid, etc.).

[0073] Neurofilament light chain (NfL) Neurofilaments are neuronal cytoskeletal components that are particularly abundant in axons. Functions of neurofilaments include providing structural support and maintaining the size, shape and internal diameter of axons. Neurofilaments contain three subunits: neurofilament light chain (NfL), neurofilament medium chain and neurofilament heavy chain. Levels of NfL increase in cerebrospinal fluid (CSF) and blood in proportion to the degree of axonal damage in various neurological disorders, including inflammatory, neurodegenerative, traumatic and cerebrovascular diseases. Although NfL has been used as a biomarker for neurodegenerative disorders, its association with other diseases and conditions, including cardiac conditions, has not been fully explored.

[0074] As described herein, in some embodiments, neurofilament light chain (NfL) may be useful for the detection and diagnosis of TTR-CM. In some embodiments, NfL is an ATTR biomarker. In some embodiments, detection of NfL, a characteristic fragment of NfL, and / or a variant of NfL is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, NfL is detected in a sample using an anti-NfL drug (e.g., an anti-NfL antibody drug, a probe, etc.). In some embodiments, detection of nucleotides encoding NfL, a characteristic fragment of NfL, and / or a variant of NfL is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, nucleotides encoding NfL are detected in a sample using an anti-NfL nucleotide sequence drug (e.g., an anti-NfL nucleotide sequence antibody drug, a probe, a complementary nucleic acid, etc.).

[0075] Decorin (DCN) Decorin (DCN) is a proteoglycan with an average molecular weight of 90-140 kilodaltons (kDa). It belongs to the small leucine-rich proteoglycan (SLRP) family and contains a protein core containing leucine repeats together with glycosaminoglycan (GAG) chains of either chondroitin sulfate (CS) or dermatan sulfate (DS). DCN is a component of connective tissue and binds to type I collagen fibrils. DCN also acts as a ligand for various cytokines and growth factors by directly or indirectly interacting with the corresponding signaling molecules involved in cell growth, differentiation, proliferation, adhesion and metastasis, DCN plays a particularly crucial role in cancer cell proliferation, dissemination, proinflammatory processes and antigen fibrillogenesis.

[0076] In some embodiments of the present disclosure, DCN is an ATTR biomarker. In some embodiments, detection of DCN, characteristic fragments of DCN and / or variants of DCN is used in a method for assessing the risk of a subject developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, DCN is detected in a sample using an anti-DCN drug (e.g., anti-DCN antibody drug, probe, etc.). In some embodiments, detection of nucleotides encoding DCN, characteristic fragments of DCN and / or variants of DCN is used in a method for assessing the risk of a subject developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, nucleotides encoding DCN are detected in a sample using an anti-DCN nucleotide sequence drug (e.g., anti-DCN nucleotide sequence antibody drug, probe, complementary nucleic acid, etc.).

[0077] SPARC-related modular calcium binding 2 (SMOC-2) SPARC-related modular calcium-binding protein 2 (SMOC-2), previously called SMAP2 (smooth muscle associated protein 2), is a 55 kDa glycoprotein that is a member of the SPARC family of matricellular proteins. SMOC-2 promotes cell cycle progression by signaling through integrin-linked kinase (ILK) to upregulate cyclin D1. When expressed in the endothelial extracellular matrix, it enhances growth factor-induced angiogenesis. SMOC-2 expression is upregulated during neointima formation and promotes vascular smooth muscle proliferation and migration. In skin, it promotes keratinocyte attachment and migration. SMOC-2 can also inhibit proteases in lungs and arteries. SMOC-2 has been proposed as a biomarker for several cancers.

[0078] In some embodiments, SPARC-related modular calcium binding 2 (SMOC2) is an ATTR biomarker. In some embodiments, detection of SMOC2, a characteristic fragment of SMOC2, and / or a variant of SMOC2 is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, SMOC2 is detected in a sample using an anti-SMOC2 drug (e.g., an anti-SMOC2 antibody drug, a probe, etc.). In some embodiments, detection of nucleotides encoding SMOC2, a characteristic fragment of SMOC2, and / or a variant of SMOC2 is used in a method for assessing a subject's risk of developing TTR-CM, diagnosing a subject with TTR-CM, or recommending a subject for further cardiomyopathy testing. In some embodiments, nucleotides encoding SMOC2 are detected in a sample using an anti-SMOC2 nucleotide sequence drug (e.g., an anti-SMOC2 nucleotide sequence antibody drug, a probe, a complementary nucleic acid, etc.).

[0079] Exemplary amino acid sequences of certain ATTR biomarkers disclosed herein are included in Table 1, below.

[0080] [Table 1-1] [Table 1-2]

[0081] In some embodiments, additional markers may be analyzed or evaluated, hi some such embodiments, the additional markers include misfolded or aggregated transthyretin (TTR).

[0082] In some embodiments, additional factors are taken into account, including, but not limited to, demographic factors of the subject from which the sample was obtained (e.g., one or more of age, weight, biological sex, ethnicity, BMI, medical history, risk factors, family history and geographic location) and / or image-based biomarkers (e.g., left ventricular septum thickness and / or ejection fraction).

[0083] Exemplary Combinations of ATTR Biomarkers As provided herein, each ATTR biomarker can be a full-length protein or a fragment thereof. In some embodiments, the fragment of the ATTR biomarker is a characteristic fragment. In some embodiments, the ATTR biomarker is a full-length ATTR biomarker protein. In some embodiments, the ATTR biomarker is a characteristic fragment of the ATTR biomarker. In some embodiments, a subset of the ATTR biomarkers is a full-length ATTR biomarker protein and a subset of the ATTR biomarkers is a characteristic fragment of the ATTR biomarker.

[0084] In some embodiments, the ATTR biomarkers have a wild-type amino acid sequence. In some embodiments, the ATTR biomarkers have a variant amino acid sequence, e.g., an amino acid sequence that includes one or more mutations. In some embodiments, the ATTR biomarkers each have a wild-type amino acid sequence. In some embodiments, the ATTR biomarkers each have a variant amino acid sequence. In some embodiments, a subset of the ATTR biomarkers each have a wild-type amino acid sequence and a subset of the ATTR biomarkers each have a variant amino acid sequence.

[0085] In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises a combination of the ATTR biomarkers listed below in Tables 2-5. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises a combination of the ATTR biomarkers listed below in Table 2. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises a combination of the ATTR biomarkers listed below in Table 3. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises a combination of the ATTR biomarkers listed below in Table 4. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises a combination of the ATTR biomarkers listed below in Table 5.

[0086] In some embodiments, the combination of ATTR biomarkers includes one or more of TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the combination of ATTR biomarkers includes two or more of TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the combination of ATTR biomarkers includes three or more of TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the combination of ATTR biomarkers includes four or more of TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the combination of ATTR biomarkers includes 5 or more of TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the combination of ATTR biomarkers includes 6 or more of TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the combination of ATTR biomarkers includes 7 or more of TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the combination of ATTR biomarkers includes 8 or more of TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the combination of ATTR biomarkers includes TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, or NfL.

[0087] In some embodiments, the combination of ATTR biomarkers comprises TnI. In some embodiments, the combination of ATTR biomarkers comprises TnI and PKM1. In some embodiments, the combination of ATTR biomarkers comprises TnI and PKM2. In some embodiments, the combination of ATTR biomarkers comprises TnI, PKM1 and PKM2. In some embodiments, the combination of ATTR biomarkers further comprises NT-proBNP, RBP4 or both. In some embodiments, the combination of ATTR biomarkers further comprises TIMP2, NfL or both.

[0088] In some embodiments, the combination of ATTR biomarkers comprises NT-proBNP. In some embodiments, the combination of ATTR biomarkers further comprises TnI, PKM1, PKM2, RBP4, or a combination thereof. In some embodiments, the combination of ATTR biomarkers further comprises TIMP2, NfL, or both.

[0089] In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises TnI, PKM2, NT-proBNP, and RBP4. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises TnI, PKM2, and NT-proBNP. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises TnI, PKM2, and RBP4. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises TnI, PKM1, and NT-proBNP. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises TnI, PKM1, and RBP4. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes TnI, PKM1, PKM2, NT-proBNP, and RBP4. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes TnI, PKM1, PKM2, and NT-proBNP. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes TnI, PKM1, PKM2, and RBP4. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes NT-proBNP, RBP4, and TnI. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises RBP4, SMOC-2, and TnI. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises DCN, NT-proBNP, and TnI. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises DCN, RBP4, and TnI.In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises NT-proBNP, SMOC-2, and TnI. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises NT-proBNP, TIMP2, and TnI. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises NT-proBNP and TnI. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises DCN and TnI. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises DCN, TIMP2, and TnI. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises RBP4 and TnI. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises RBP4, TIMP2 and TnI. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises DCN, SMOC-2 and TnI. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises TIMP2 and TnI. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) comprises SMOC-2, TIMP2 and TnI.

[0090] Exemplary combinations of ATTR biomarkers consistent with the present disclosure are included below in Table 2. Also disclosed herein are combinations of ATTR biomarkers, including those listed in Table 2 below.

[0091] [Table 2]

[0092] Exemplary combinations comprising two ATTR biomarkers consistent with the present disclosure are included below in Table 3. Also disclosed herein are combinations of ATTR biomarkers, including those listed in Table 3 below.

[0093] [Table 3]

[0094] Exemplary combinations comprising three ATTR biomarkers consistent with the present disclosure are included below in Table 4. Also disclosed herein are combinations of ATTR biomarkers, including those listed in Table 4 below.

[0095] [Table 4-1] [Table 4-2] [Table 4-3]

[0096] Exemplary combinations including four ATTR biomarkers consistent with the present disclosure are included below in Table 5. Also disclosed herein are combinations of ATTR biomarkers, including the combinations listed in Table 5 below.

[0097] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0098] In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) does not include PKM1. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) does not include PKM2. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) does not include PKM or PKM2.

[0099] In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) does not include SMOC-2. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) does not include DCN. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) does not include SMOC-2 or DCN.

[0100] method The disclosed method further allows for early identification of more patients at risk for TTR-CM while minimizing the number of false negatives. In some embodiments, the disclosed method provides the advantage of early screening of the presence of ATTRwt. In some embodiments, the disclosed method can assist in the detection or diagnosis of ATTRwt after genetic testing has ruled out ATTRm. In some embodiments, the disclosed method reduces or eliminates the need to begin screening for TTR-CM with an echocardiogram, followed by CMR and scintigraphy, which is a costly and complicated process. In some embodiments, if the disclosed method detects possible TTR-CM in a subject, the subject can undergo subsequent confirmatory testing, such as an echocardiogram, cardiac magnetic resonance imaging (CMR), scintigraphy and / or cardiac biopsy.

[0101] In some embodiments, the methods disclosed herein are methods of determining a subject's risk of developing amyloid transthyretin cardiomyopathy (TTR-CM). In some embodiments, the methods disclosed herein are methods of diagnosing a subject with TTR-CM, and a sample is obtained from the subject. In some embodiments, the methods disclosed herein are methods of treating TTR-CM in a subject at risk for or suffering from TTR-CM. In some embodiments, the methods disclosed herein are methods of determining that a patient does not have TTR-CM or is not at risk for developing TTR-CM.

[0102] In some embodiments, the TTR-CM results from wild-type transthyretin amyloidosis (ATTRwt). In some embodiments, the subject tests negative for familial amyloidotic cardiomyopathy (ATTRm) by genetic testing.

[0103] In some embodiments, the methods disclosed herein are methods of selecting a subject to receive one or more doses of a TTR stabilizer, and the sample is obtained from the subject. In some embodiments, the methods disclosed herein include administering one or more doses of a TTR stabilizer to the subject.

[0104] In some embodiments, the method disclosed herein is a method of selecting a subject for one or more cardiomyopathy tests, and a sample is obtained from the subject. In some embodiments, the one or more cardiomyopathy tests include an echocardiogram, an advanced imaging method, or both. In some embodiments, the advanced imaging method includes cardiac magnetic resonance imaging (CMR), scintigraphy, or both. In some embodiments, the scintigraphy includes the use of a radioisotope conjugate, such as 99m Tc-pyrophosphate. In some embodiments, the scintigraphy is performed using single photon emission computed tomography (SPECT).

[0105] The present disclosure provides diagnostic tests for TTR-CM (including TTR-CM caused by wild-type ATTR amyloidosis) that feature detection of biomarkers by the methods described above.

[0106] In some embodiments, the methods of detecting, diagnosing or identifying risk of TTR-CM as taught by the present disclosure are improved methods in that, compared to standard techniques, the methods of the present disclosure include one or more of the following benefits: improved sensitivity for identifying TTR-CM, improved specificity for identifying TTR-CM, improved accuracy for identifying TTR-CM, reduced time to diagnosis of TTR-CM, and / or reduced costs of screening patients for TTR-CM.

[0107] In some embodiments, the biomarkers disclosed herein can be used in in vitro diagnostic (IVD) or screening tests for amyloid transthyretin cardiomyopathy conditions. In some embodiments, the diagnostic tests taught by the present disclosure detect whether one or more biomarkers are present in a sample obtained from a subject. In some embodiments, the diagnostic tests taught by the present disclosure can aid in the detection or diagnosis of TTR-CM (including TTR-CM caused by wild-type ATTR amyloidosis) in a subject.

[0108] In some embodiments, the diagnostic tests taught by the present disclosure are suitable for immunoassay platforms. In some embodiments, such immunoassay platforms include semi-automated or automated immunoassay platforms. In some embodiments, the diagnostic tests taught by the present disclosure are suitable for semi-automated testing of one or more biomarkers.

[0109] In some embodiments, the diagnostic tests taught by the present disclosure are improved diagnosis of TTR-CM, in that, compared to standard techniques, the diagnostic tests of the present disclosure include one or more of the following advantages: improved sensitivity for identifying TTR-CM, improved specificity for identifying TTR-CM, improved accuracy for identifying TTR-CM, reduced time to diagnosis of TTR-CM, and / or reduced costs of screening patients for TTR-CM.

[0110] In some embodiments, the diagnostic test disclosed herein may be a plasma-based screening assay. In some embodiments, the diagnostic test is suitable for, for example, a Siemens Atellica® system or a Siemens Advia Centaur® system.

[0111] As described in more detail below, in some embodiments, the methods provided herein comprise detecting the level of each of two or more transthyretin amyloidosis (ATTR) biomarkers present in a sample.

[0112] In some embodiments, the method provided herein comprises detecting the level of each of two or more transthyretin amyloidosis (ATTR) biomarkers in a sample to obtain an ATTR biomarker profile, and using the ATTR biomarker profile to compute an ATTR biomarker score. In some embodiments, the method provided herein comprises detecting the level of each of two or more transthyretin amyloidosis (ATTR) biomarkers in a sample to obtain an ATTR biomarker profile, and using the ATTR biomarker profile and demographic factors to compute an ATTR biomarker score. In some embodiments, the method provided herein comprises detecting the level of each of two or more transthyretin amyloidosis (ATTR) biomarkers in a sample to obtain an ATTR biomarker profile, and using the ATTR biomarker profile and image-based biomarkers to compute an ATTR biomarker score. In some embodiments, the methods provided herein include detecting a level of each of two or more transthyretin amyloidosis (ATTR) biomarkers in a sample to obtain an ATTR biomarker profile, and computing an ATTR biomarker score using the ATTR biomarker profile, demographic factors, and image-based biomarkers.

[0113] In some embodiments, the methods provided herein include receiving a level of each of two or more transthyretin amyloidosis (ATTR) biomarkers in a sample. In some embodiments, the receiving includes receiving electronically.

[0114] In some embodiments, the method provided herein comprises using the respective levels of two or more transthyretin amyloidosis (ATTR) biomarkers in a sample to obtain an ATTR biomarker profile, and using the ATTR biomarker profile to compute an ATTR biomarker score. In some embodiments, the method provided herein comprises using the respective levels of two or more transthyretin amyloidosis (ATTR) biomarkers in a sample to obtain an ATTR biomarker profile, and using the ATTR biomarker profile and demographic factors to compute an ATTR biomarker score. In some embodiments, the method provided herein comprises using the respective levels of two or more transthyretin amyloidosis (ATTR) biomarkers in a sample to obtain an ATTR biomarker profile, and using the ATTR biomarker profile and image-based biomarkers to compute an ATTR biomarker score. In some embodiments, the methods provided herein include using the levels of each of two or more transthyretin amyloidosis (ATTR) biomarkers in the sample to obtain an ATTR biomarker profile, and using the ATTR biomarker profile, demographic factors, and image-based biomarkers to compute an ATTR biomarker score.

[0115] In some embodiments, the demographic factors include one or more of age, weight, biological sex, ethnicity, BMI, medical history, risk factors, family history, and geographic location. In some embodiments, the image-based biomarkers include left ventricular septum thickness and / or ejection fraction.

[0116] In some embodiments, the methods described herein include using the score of the ATTR biomarkers to select a subject for further cardiomyopathy testing. In some embodiments, the methods described herein include using the score of the ATTR biomarkers to select a subject for receiving one or more doses of a TTR stabilizer. In some embodiments, the methods described herein include using the score of the ATTR biomarkers to identify a subject as having or at risk of having TTR-CM.

[0117] In some embodiments, the methods described herein include comparing the score of the ATTR biomarker to a score of a reference ATTR biomarker. In some embodiments, the methods described herein include administering one or more doses of a TTR stabilizer to the subject. In some embodiments, the methods described herein include performing one or more cardiomyopathy tests on the subject.

[0118] Further description of the exemplary methods disclosed herein is provided below.

[0119] Detection of ATTR biomarkers and acquisition of ATTR biomarker profiles Among other things, the method provided herein comprises evaluating the level of one or more ATTR biomarkers in a sample.The level of ATTR biomarkers can be detected in a sample.The exemplary method for detecting the level of one or more ATTR biomarkers is described herein.However, the level of ATTR biomarkers can also be provided, for example, in electronic form, for example, by the laboratory that detects the level of one or more ATTR biomarkers in a sample.

[0120] In some embodiments, the present disclosure provides a technique in which one or more ATTR biomarkers are detected, analyzed and / or evaluated in a sample.In some embodiments, one or more ATTR biomarkers are in a sample obtained from a subject; in some embodiments, a diagnostic or therapeutic decision is made based on such detection, analysis and / or evaluation.In some embodiments, the biomarkers detected, analyzed or evaluated are one or more ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or combinations thereof).

[0121] In some embodiments, the present disclosure provides a method for detecting the level of one or more ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof) in a sample. The level of an ATTR biomarker described herein encompasses the presence of an ATTR biomarker, the absence of an ATTR biomarker, the amount of an ATTR biomarker, the absolute amount of an ATTR biomarker, the relative amount of an ATTR biomarker, or the concentration of an ATTR biomarker.

[0122] In some embodiments, the methods provided herein comprise detecting in a sample the level of each ATTR biomarker in a combination listed in Tables 2-5. In some embodiments, the methods provided herein comprise detecting in a sample the level of each ATTR biomarker in a combination listed in Table 2. In some embodiments, the methods provided herein comprise detecting in a sample the level of each ATTR biomarker in a combination listed in Table 3. In some embodiments, the methods provided herein comprise detecting in a sample the level of each ATTR biomarker in a combination listed in Table 4. In some embodiments, the methods provided herein comprise detecting in a sample the level of each ATTR biomarker in a combination listed in Table 5.

[0123] In some embodiments, the methods provided herein include detecting a level of each of one or more ATTR biomarkers in a sample, where the one or more ATTR biomarkers include TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the methods provided herein include detecting a level of each of two or more ATTR biomarkers in a sample, where the two or more ATTR biomarkers include TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the methods provided herein include detecting a level of each of three or more ATTR biomarkers in a sample, where the three or more ATTR biomarkers include TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the methods provided herein include detecting the level of each of four or more ATTR biomarkers in a sample, the four or more ATTR biomarkers include TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the methods provided herein include detecting the level of each of five or more ATTR biomarkers in a sample, the five or more ATTR biomarkers include TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the methods provided herein include detecting the level of each of six or more ATTR biomarkers in a sample, the six or more ATTR biomarkers include TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof.In some embodiments, the methods provided herein include detecting the levels of each of seven or more ATTR biomarkers in a sample, the seven or more ATTR biomarkers including TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, and NfL. In some embodiments, the methods provided herein include detecting the levels of each of eight or more ATTR biomarkers in a sample, the eight or more ATTR biomarkers including TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, and NfL. In some embodiments, the methods provided herein include detecting the levels of each of TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, and NfL in a sample.

[0124] In some embodiments, the methods provided herein include detecting the level of TnI in a sample. In some embodiments, the methods provided herein include detecting the level of TnI and PKM1 in a sample. In some embodiments, the methods provided herein include detecting the level of TnI and PKM2 in a sample. In some embodiments, the methods provided herein include detecting the level of TnI, PKM1 and PKM2 in a sample. In some embodiments, the methods provided herein further include detecting the level of NT-proBNP, RBP4 or both in a sample. In some embodiments, the methods provided herein further include detecting the level of TIMP2, NfL or both in a sample.

[0125] In some embodiments, the method provided herein comprises detecting the level of NT-proBNP in the sample.In some embodiments, the method provided herein further comprises detecting the level of TnI, PKM1, PKM2, RBP4 or a combination thereof in the sample.In some embodiments, the method provided herein further comprises detecting the level of TIMP2, NfL or both in the sample.

[0126] In some embodiments, the methods provided herein include detecting the levels of TnI, PKM2, NT-proBNP, and RBP4 in a sample. In some embodiments, the methods provided herein include detecting the levels of TnI, PKM2, and NT-proBNP in a sample. In some embodiments, the methods provided herein include detecting the levels of TnI, PKM2, and RBP4 in a sample. In some embodiments, the methods provided herein include detecting the levels of TnI, PKM1, and NT-proBNP in a sample. In some embodiments, the methods provided herein include detecting the levels of TnI, PKM1, and RBP4 in a sample. In some embodiments, the methods provided herein include detecting the levels of TnI, PKM1, PKM2, NT-proBNP, and RBP4 in a sample. In some embodiments, the methods provided herein include detecting the levels of TnI, PKM1, PKM2, NT-proBNP, and RBP4 in a sample. In some embodiments, the methods provided herein include detecting the levels of TnI, PKM1, PKM2, NT-proBNP, and RBP4 in a sample. In some embodiments, the methods provided herein include detecting the levels of TnI, PKM1, PKM2, and RBP4 in a sample. In some embodiments, the methods provided herein include detecting the levels of NT-proBNP, RBP4, and TnI in a sample. In some embodiments, the methods provided herein include detecting the levels of RBP4, SMOC-2, and TnI in a sample. In some embodiments, the methods provided herein include detecting the levels of DCN, NT-proBNP, and TnI in a sample. In some embodiments, the methods provided herein include detecting the levels of DCN, RBP4, and TnI in a sample. In some embodiments, the methods provided herein include detecting the levels of NT-proBNP, SMOC-2, and TnI in a sample. In some embodiments, the methods provided herein include detecting the levels of NT-proBNP, TIMP2, and TnI in a sample.In some embodiments, the methods provided herein include detecting the respective levels of NT-proBNP and TnI in a sample. In some embodiments, the methods provided herein include detecting the respective levels of DCN and TnI in a sample. In some embodiments, the methods provided herein include detecting the respective levels of DCN, TIMP2, and TnI in a sample. In some embodiments, the methods provided herein include detecting the respective levels of RBP4 and TnI in a sample. In some embodiments, the methods provided herein include detecting the respective levels of RBP4, TIMP2, and TnI in a sample. In some embodiments, the methods provided herein include detecting the respective levels of DCN, SMOC-2, and TnI in a sample. In some embodiments, the methods provided herein include detecting the respective levels of TIMP2 and TnI in a sample. In some embodiments, the methods provided herein include detecting the respective levels of SMOC-2, TIMP2, and TnI in a sample.

[0127] Methods for detecting a biomarker (e.g., an ATTR biomarker) include methods for detecting the biomarker as a protein. Protein-based biomarker detection methods include, for example, mass spectrometry (MS), immunoassays (e.g., immunoprecipitation), Western blot, ELISA, immunohistochemistry, immunocytochemistry, flow cytometry, and / or immuno-PCR.

[0128] In some embodiments, mass spectrometry is performed by MS, MS / MS, MALDI-TOF, electrospray ionization mass spectrometry (ESIMS), ESI-MS / MS, ESI-MS / (MS) n, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS), tandem liquid chromatography mass spectrometry (LC-MS / MS) mass spectrometry, desorption / ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS / MS, APCI-(MS), atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS and APPI-(MS) n Mass spectrometry includes quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS) and ion trap mass spectrometry. In many cases, MS approaches quantify fragments of biomarkers rather than full-length proteins. However, MS approaches may be sufficient to determine protein levels of biomarkers to an accuracy sufficient for the disclosed methods and / or evaluations.

[0129] In some embodiments, the immunoassay can be a chemiluminescence immunoassay.In some embodiments, the immunoassay can be a high-throughput and / or automated immunoassay platform.For example, the high-throughput and / or automated immunoassay platform can be used to analyze at least 240 tests per hour or at least 440 tests per hour.

[0130] In some embodiments, a method for detecting a biomarker as a protein in a sample comprises contacting the sample with one or more antibody drugs against the biomarker of interest. In some embodiments, such a method also comprises contacting the sample with a first set of one or more detection drugs. In some embodiments, the antibody drugs are labeled with a first set of one or more detection drugs. In some embodiments, the first set of one or more detection drugs comprises one or more acridinium ester molecules.

[0131] Acridinium ester (AE) molecules can be used to label proteins and nucleic acids. Acridinium-labeled proteins can be used for detection in immunoassays. Exposure of AE to alkaline H2O2 (hydrogen peroxide) generates chemiluminescence. Light is emitted with a maximum wavelength ranging from 430 to 480 nm, depending on the particular AE variant. Such light can be detected, for example, by a highly efficient photomultiplier tube. Light emission is rapid, lasting from 1 to 5 seconds. The diversity of AE forms contributes to better assay performance, including improved sensitivity and robustness. AE molecules can be used to label small molecules, large analytes, and antibodies.

[0132] Further methods of detecting biomarkers include methods for detecting biomarkers as nucleic acids. Nucleic acid-based biomarker detection methods include performing nucleic acid amplification methods, such as polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-specific amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA). In some embodiments, nucleic acid-based biomarker detection methods include detecting hybridization between one or more nucleic acid probes and one or more nucleotides encoding the biomarker of interest. In some embodiments, the nucleic acid probes are each complementary to at least a portion of one of the one or more nucleotides encoding the biomarker of interest. In some embodiments, the nucleotides encoding the biomarker of interest comprise DNA (e.g., cDNA). In some embodiments, the nucleotides encoding the biomarker of interest comprise RNA (e.g., mRNA).

[0133] In some embodiments, the methods provided herein detect the levels of one or more ATTR biomarkers (e.g., two or more, three or more, four or more, or five or more ATTR biomarkers) to obtain an ATTR biomarker profile. In some embodiments, the ATTR biomarker profile includes the levels of each of the ATTR biomarkers that are evaluated. In some embodiments, the ATTR biomarker profile includes the levels of each of the ATTR biomarkers that are detected, e.g., as described herein.

[0134] sample In some embodiments, the sample disclosed herein is a biological sample.In some embodiments, the biological sample is a blood sample, for example, drawn from a subject's artery or vein.The blood sample can be a whole blood sample, a plasma sample or a serum sample.In some embodiments, the biological sample comprises heart tissue.

[0135] In some embodiments, the sample is obtained from a subject. In some embodiments, the subject from whom the sample was obtained has been evaluated for TTR-CM. In some embodiments, the subject from whom the sample was obtained has or is at risk for developing amyloid transthyretin cardiomyopathy (TTR-CM).

[0136] In some embodiments, the methods disclosed herein include obtaining a biological sample from a subject. In some embodiments, obtaining a biological sample from a subject includes drawing blood. In some embodiments, obtaining a biological sample from a subject includes performing a biopsy.

[0137] In some embodiments of the methods disclosed herein, the sample is provided, for example, by a medical professional.

[0138] subject In some embodiments, the subject disclosed herein is a mammal. In some embodiments, the mammal is a human.

[0139] In some embodiments, the subject disclosed herein is a biological male. In some embodiments, the subject disclosed herein is a biological female.

[0140] In some embodiments, the subject disclosed herein is overweight. In some embodiments, the subject has a body mass index (BMI) of 25 or more. In some embodiments, the subject has a body mass index (BMI) of 30 or more.

[0141] In some embodiments, the subject is at least 50 years old. In some embodiments, the subject is at least 55 years old. In some embodiments, the subject is at least 60 years old. In some embodiments, the subject is at least 65 years old.

[0142] How to use thresholds In some methods disclosed herein, the level of one or more ATTR biomarkers can be compared to a threshold value.In some embodiments, the method disclosed herein comprises comparing the level of one or more ATTR biomarkers to a respective threshold value.In some embodiments, the method disclosed herein comprises comparing the level of one or more ATTR biomarkers to a reference threshold value.

[0143] The reference threshold value may be a threshold value from a subject known or independently verified to have good cardiac health, or from a subject known or independently verified to have poor cardiac health, such as in the case of a subject with TTR-CM.Alternatively, or in combination, the subject's ATTR biomarker profile is compared to a reference threshold value determined from multiple subjects with a common known condition (e.g., healthy, not diagnosed with TTR-CM, or diagnosed with TTR-CM).In some embodiments, the reference threshold value is the average of the known levels of the ATTR biomarker from multiple subjects, or alternatively, is a range defined by the range of levels of the ATTR biomarker observed in the reference subjects.

[0144] In more complex assessment approaches, the subject's ATTR biomarker levels are compared to reference ATTR biomarker levels constructed from a larger number of subjects with a common condition (e.g., healthy, not diagnosed with TTR-CM, or diagnosed with TTR-CM), e.g., at least 10, at least 50, at least 100, at least 500, at least 1000, or more. In many cases, the reference subjects have an even distribution of conditions between (1) being healthy / not diagnosed with TTR-CM and (2) being diagnosed with TTR-CM. In some cases, the assessment involves repeated or simultaneous comparison of the subject's ATTR biomarker levels to multiple profiles of known conditions.

[0145] A plurality of known reference ATTR biomarker profiles (e.g., detection levels of one or more ATTR biomarkers in a reference sample) can also be used to train a computational assessment algorithm, e.g., a machine learning model, such that a single comparison between a subject's ATTR biomarker profile and the reference ATTR biomarker profile provides a result that integrates or aggregates information from a large number of subjects, e.g., at least 10, at least 50, at least 100, at least 500, at least 1000 or more individuals, with a common known health condition (e.g., healthy, not diagnosed with TTR-CM, or diagnosed with TTR-CM). The generation of such a reference ATTR biomarker profile can facilitate a faster assessment of a subject's risk for TTR-CM, or an assessment using less computational power.

[0146] Reference ATTR biomarker profile can be generated from multiple reference ATTR biomarker profiles by any of several computational approaches known to those skilled in the art.Machine learning model can be easily constructed, for example, using any number of statistical programming languages, such as R, scripting languages, such as Python, and related machine learning packages, data mining software, such as Weka or Java, Mathematica, Matlab or SAS.

[0147] The subject's ATTR biomarker profile can be compared with a reference ATTR biomarker profile generated as described above or otherwise by a person skilled in the art to generate an output assessment. Some output assessments are consistent with the present disclosure herein. The output assessment includes a single assessment, often refined by sensitivity, specificity, or sensitivity and specificity parameters, and indicates a health status assessment (e.g., the probability that the subject has TTR-CM, the subject is not at risk for TTR-CM, the subject is at risk for TTR-CM, the subject has TTR-CM). Alternately or in combination, additional parameters are provided, such as the subject's demographic factors (e.g., one or more of age, weight, biological sex, ethnicity, BMI, medical history, risk factors, family history, and geographic location) and / or the subject's image-based biomarkers (e.g., left ventricular septum thickness and / or ejection fraction).

[0148] More specifically, in some embodiments, the methods disclosed herein further comprise diagnosing the subject with amyloid transthyretin cardiomyopathy (TTR-CM) when the level of at least one of the detected one or more ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof) is above a threshold value. In some embodiments, the methods disclosed herein further comprise diagnosing the subject with amyloid transthyretin cardiomyopathy (TTR-CM) when the level of at least one of the detected one or more ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof) is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, or at least 1.9 times greater than the threshold value. In some embodiments, the methods disclosed herein further comprise diagnosing the subject with amyloid transthyretin cardiomyopathy (TTR-CM) if the level of each of the one or more ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof) detected is above a threshold value. In some embodiments, the methods disclosed herein further comprise diagnosing the subject with amyloid transthyretin cardiomyopathy (TTR-CM) if the level of each of the one or more ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof) detected is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, or at least 1.9 times greater than the threshold value.

[0149] In some embodiments, the methods disclosed herein further comprise recommending the subject to one or more cardiomyopathy tests when the level of at least one or more ATTR biomarkers is above a threshold value. In some such methods, the subject is recommended to one or more cardiomyopathy tests when the level of at least one of the detected one or more ATTR biomarkers is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8 or at least 1.9 times greater than the threshold value. In some embodiments, the methods disclosed herein further comprise recommending the subject to one or more cardiomyopathy tests when the level of each of the one or more ATTR biomarkers is above a threshold value. In some such methods, the subject is recommended to one or more cardiomyopathy tests when the level of each of the detected one or more ATTR biomarkers is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8 or at least 1.9 times greater than the threshold value.

[0150] As discussed above, in some embodiments, a method for detecting one or more ATTR biomarkers (e.g., ATTR biomarker proteins) in a sample comprises contacting the sample with one or more antibody drugs against the ATTR biomarkers. In some embodiments, such a method also comprises contacting the sample with a first set of one or more detection agents. In some embodiments, the antibody drugs are labeled with the first set of one or more detection agents. In some embodiments, the first set of one or more detection agents comprises one or more acridinium ester molecules.

[0151] Acridinium ester (AE) molecules can be used to label proteins and nucleic acids. Acridinium-labeled proteins can be used for detection in immunoassays. Exposure of AE to alkaline H2O2 (hydrogen peroxide) produces chemiluminescence. Light is emitted with a maximum wavelength in the range of 430-480 nm, depending on the particular AE variant. Such light can be detected, for example, by a highly efficient photomultiplier tube.

[0152] In some embodiments, detecting binding between the ATTR biomarker and one or more antibody drugs against the ATTR biomarker comprises determining an absorbance or luminescence value for a first set of one or more detection agents. For example, the absorbance value indicates the level of binding (e.g., higher absorbance indicates more binding). In some embodiments, the absorbance or luminescence value for the first set of one or more detection agents is above a threshold value. In some embodiments, the absorbance or luminescence value for the first set of one or more detection agents is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, or at least 1.9 times greater than the threshold value. In some embodiments, the threshold value is the average of the absorbance or luminescence values ​​determined for a second set of one or more detection agents labeling two or more control samples. In some such embodiments, the second set of one or more detection agents is similar or the same as the first set of one or more detection agents.

[0153] In one aspect, the present disclosure provides a method for detecting one or more ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof) in a subject, wherein the method for detecting biological markers described above is followed to detect whether one or more ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof) are present in a sample obtained from the subject. In some embodiments, the method for detecting one or more ATTR biomarkers comprises detecting binding between an ATTR biomarker and one or more anti-ATTR biomarker antibody drugs. In some embodiments, the step of detecting whether one or more ATTR biomarkers are present in a sample obtained from the subject comprises detecting the level of one or more ATTR biomarkers present in the sample obtained from the subject. In some embodiments, the level of the detected ATTR biomarker is above a threshold value for each ATTR biomarker. In some embodiments, the level of the ATTR biomarker detected is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, or at least 1.9 times greater than the threshold value.

[0154] In some embodiments, the present disclosure provides a method for detecting one or more ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof) in a sample obtained from a subject. Biological markers for amyloid transthyretin cardiomyopathy can include TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the control sample used in the above method comprises a sample obtained from one or more subjects who do not have ATTR amyloidosis and / or TTR-CM.

[0155] In some embodiments, the methods disclosed herein further comprise diagnosing the subject with amyloid transthyretin cardiomyopathy (TTR-CM) if the level of at least one or one or more detected ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof) is above a threshold. In some embodiments, the methods comprise diagnosing the subject with TTR-CM if the level of at least one or one or more detected ATTR biomarkers is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, or at least 1.9 times greater than the threshold.

[0156] In some embodiments, the methods disclosed herein further comprise diagnosing the subject with amyloid transthyretin cardiomyopathy (TTR-CM) if the absorbance or luminescence value for the first set of one or more detection agents is greater than a threshold value. In some embodiments, the method comprises diagnosing the subject with TTR-CM if the absorbance or luminescence value for the first set of one or more detection agents is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, or at least 1.9 times greater than the threshold value. In some embodiments, the threshold value is the average of the absorbance or luminescence value determined for a second set of one or more detection agents labeling two or more control samples. In some such embodiments, the second set of one or more detection agents is similar or the same as the first set of one or more detection agents.

[0157] In some embodiments, the methods disclosed herein further comprise recommending the subject to one or more cardiomyopathy tests when the level of at least one or one or more ATTR biomarkers is above a threshold value.In some such methods, the subject is recommended to one or more cardiomyopathy tests when the level of at least one or one or more detected ATTR biomarkers is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8 or at least 1.9 times greater than the threshold value.

[0158] In some embodiments, the methods disclosed herein further comprise recommending the subject for one or more cardiomyopathy tests if the absorbance or luminescence value for the first set of one or more detection agents is greater than a threshold value. In some such methods, the subject is recommended for one or more cardiomyopathy tests if the absorbance or luminescence value for the first set of one or more detection agents is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, or at least 1.9 times greater than the threshold value.

[0159] Cardiomyopathy tests that can be used in accordance with the methods of the present disclosure include echocardiograms or advanced imaging methods. In some embodiments, advanced imaging methods include cardiac magnetic resonance imaging (CMR) or scintigraphy (e.g., 99m(using a radioisotope conjugate such as Tc-pyrophosphate, and / or using single photon emission computed tomography (SPECT). In some embodiments, the methods disclosed herein further comprise recommending the subject for cardiac biopsy if the level of at least one or one or more detected ATTR biomarkers is above a threshold and the subject tests positive for cardiomyopathy in one or more of the cardiomyopathy tests. In some such embodiments, the biopsy tissue is tested for cardiomyopathy by immunochemical staining. For example, the immunochemical staining includes the use of one or more antibody drugs against kappa or lambda light chain amyloid deposits in cardiac tissue, and / or one or more antibody drugs against transthyretin deposits. If the immunochemical staining shows the presence of transthyretin deposits in cardiac tissue, the subject can be diagnosed with amyloid transthyretin cardiomyopathy (TTR-CM).

[0160] In any of the above embodiments, the threshold value may be the average of the values ​​detected for two or more control samples. In some such embodiments, the values ​​detected for two or more control samples represent control levels for one or more ATTR biomarkers. In some embodiments, the control samples include recombinant ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or combinations thereof). In some embodiments, each of the two or more control samples is a sample obtained from a subject without TTR-CM. In some embodiments, the threshold value is a value reported in a standard table.

[0161] How to Use ATTR Biomarker Profiles The algorithm-based assay and related information provided by implementing any of the methods described herein can facilitate optimal treatment and decision-making for a subject.For example, the methods described herein can allow a physician or caregiver to identify patients who are unlikely to have TTR-CM and therefore will not require treatment, will not require further cardiac testing, or will not require increased monitoring for TTR-CM, or patients who are likely to have TTR-CM and will require treatment, will require further cardiac testing, or will require increased monitoring for TTR-CM.

[0162] The score of ATTR biomarkers can be determined by applying a specific algorithm in some cases. In some embodiments, the score of ATTR biomarkers is quantitative. The algorithm used to calculate the score of ATTR biomarkers in the methods disclosed herein can group the expression level values ​​of ATTR biomarkers or ATTR biomarker groups. Furthermore, forming a specific group of ATTR biomarkers can facilitate the mathematical weighting of the various expression level contributions of ATTR biomarkers or ATTR biomarker subsets (e.g., classifiers) to the quantitative score.

[0163] Exemplary ATTR biomarkers and corresponding amino acid sequences are listed in Table 1. Exemplary combinations of ATTR biomarkers are listed in Tables 2-5.

[0164] The methods described herein and the kits and systems provided herein can utilize algorithm-based diagnostic assays to predict whether a subject from whom a sample was obtained is at risk for or afflicted with TTR-CM, to select a subject from whom a sample was obtained for one or more cardiomyopathy tests, and / or to select a subject from whom a sample was obtained to receive one or more doses of a TTR stabilizer.

[0165] The levels of one or more ATTR biomarkers and optionally one or more demographic factors (e.g., one or more of age, weight, biological sex, ethnicity, BMI, medical history, risk factors, family history, and geographic location) and / or image-based biomarkers (e.g., left ventricular septum thickness and / or ejection fraction) can be used alone or arranged into functional subsets to calculate an ATTR biomarker score that is used to predict whether the subject from whom the sample was obtained is at risk for or has TTR-CM, to select the subject from whom the sample was obtained for one or more cardiomyopathy tests, and / or to select the subject from whom the sample was obtained to receive one or more doses of a TTR stabilizer.

[0166] The method disclosed herein comprises using the ATTR biomarker profile to compute the score of ATTR biomarkers.In some embodiments, using the ATTR biomarker profile to compute the score of ATTR biomarkers comprises applying an algorithm to the ATTR biomarker profile to compute the score of ATTR biomarkers.In some embodiments, the algorithm is or is derived from a decision tree method, a neural boosting method, a bootstrap forest method, a boosted tree method, a K nearest neighbor method, a generalized regression forward variable method, a generalized regression pruning forward variable method, a fit stepwise method, a generalized regression lasso method, a generalized regression elastic net method, a generalized regression ridge method, a nominal logistic method, a support vector machine method, a discriminant method, a naive Bayes method, or a combination thereof. In some embodiments, the algorithm is or is derived from a decision tree method, a neural boosting method, a bootstrap forest method, a boosted tree method, a generalized regression lasso method, a generalized regression elastic net method, a generalized regression ridge method, a nominal logistic method, a support vector machine method, a discriminant method, or a combination thereof. In some embodiments, the algorithm is or is derived from a decision tree method, a neural boosting method, a bootstrap forest method, a boosted tree method, a support vector machine method, or a combination thereof.

[0167] Further algorithms can be used in the methods provided herein, and the algorithms provided above are merely illustrative of the types of algorithms that can be used to generate ATTR biomarker scores.Exemplary algorithms are described, for example, in Duda, 2001, Pattern Classification, John Wiley & Sons, Inc., New York. pp. 396-408 and 411-412; and Hastie et al., 2001, The Elements of Statistical Learning, Springer-Verlag, New York, Chapter 9, each of which is incorporated herein by reference.In addition, as mentioned above, combinations of algorithms can be used in the methods provided herein.For example, boosted tree method can be a combination of decision tree method and boosting method.Further combinations are possible and are contemplated for use in the methods provided herein.Exemplary algorithms that can be used in the methods provided herein are described below.

[0168] Decision Tree One methodology that can be used to calculate the score of ATTR biomarker from ATTR biomarker profile is decision tree.Decision tree can be constructed using training population and specific data analysis algorithm.Decision tree is generally described in Duda, 2001, Pattern Classification, John Wiley & Sons, Inc., New York. pp. 395-396, which is incorporated herein by reference.Tree-based methods divide feature space into a series of rectangles, and then fit a model (such as a constant) to each rectangle.

[0169] The training population data can include ATTR biomarker profiles (e.g., including the levels of one or more ATTR biomarkers in samples) across the entire training set population. One particular algorithm that can be used to build a decision tree is classification and regression tree (CART). Other particular decision tree algorithms include, but are not limited to, ID3, C4.5, MART and random forest. CART, ID3 and C4.5 are described in Duda, 2001, Pattern Classification, John Wiley & Sons, Inc., New York. pp. 396-408 and 411-412, which are incorporated herein by reference. CART, MART and C4.5 are described in Hastie et al., 2001, The Elements of Statistical Learning, Springer-Verlag, New York, Chapter 9, which is incorporated herein by reference in its entirety. Random forests are described in Breiman, 1999, "Random Forests - Random Features", Technical Report 567, Statistics Department, UC Berkeley, September 1999, the entire contents of which are incorporated herein by reference.

[0170] The purpose of a decision tree is to induce a classifier (tree) from real-world example data. This tree can be used to classify unseen examples that were not used to derive the decision tree. Thus, a decision tree can be derived from training data. Exemplary training data includes data for a plurality of subjects (e.g., a training population). An ATTR biomarker profile can be provided and / or used for each respective subject. In some embodiments, the training data includes ATTR biomarkers that proles the training population.

[0171] The following algorithm describes an exemplary decision tree derivation: Trees (e.g. classes, features) Create a root node If all examples have the same class value, give the root this label Otherwise, if the feature is empty, label the route according to its most common value. Otherwise, start the following: Calculate the information gain for each feature Select feature A with the highest information gain and make it the root feature. For each possible value of this feature, v, Add a new branch corresponding to A=v below the root Let (v) be an example with A=v. If example(v) is empty, make the new branch a leaf node labeled with the most common value in examples. Otherwise, let the new branch be the tree created by Tree(Example(v), Class, Feature-{A}) end

[0172] In a univariate decision tree, each split is based on a feature value (e.g., level) for the corresponding biomarker. Additionally, multivariate decision trees can be implemented in the manner described herein. Multivariate decision trees are described in Duda, 2001, Pattern Classification, John Wiley & Sons, Inc., New York, pp. 408-409, which is incorporated herein by reference. In such multivariate decision trees, some or all of the decisions include a linear combination of feature values ​​(e.g., levels) for multiple ATTR biomarkers in the ATTR profile. Such linear combinations can be trained using known techniques such as gradient descent in classification or by using a sum-squared-error criterion.

[0173] As an illustrative example, the following formula is used: 0.05(X1)+0.2(X2)<500

[0174] In this example, X1 and X2 refer to two different features (e.g., levels) for two different ATTR biomarkers. To apply this methodology, the values ​​of features X1 and X2 (e.g., as part of an ATTR biomarker profile) are obtained from measurements obtained from unclassified subjects. These values ​​are then inserted into the formula. If a value less than 500 is computed, then the first branch of the decision tree is selected. Otherwise, the second branch of the decision tree is selected.

[0175] To improve weak decision rules, bagging, boosting and additive trees can be combined with decision methodologies. These techniques are designed for and typically applied to decision trees such as those described above. Furthermore, such techniques may also be useful for decision rules developed using other types of data analysis algorithms, such as linear discriminant analysis.

[0176] In bagging, the training set is sampled, generating random independent bootstrap replicates, constructing a decision rule based on each of these, and aggregating these by simple majority voting in the final decision rule. See, e.g., Breiman, 1996, Machine Learning 24, 123-140; and Efron & Tibshirani, An Introduction to Boostrap, Chapman & Hall, New York, 1993, the entire contents of which are incorporated herein by reference.

[0177] In boosting, a decision rule is constructed on a weighted version of a training set that depends on previous classification results. Initially, all features under consideration have equal weights, and a first decision rule is constructed on this data set. Then, the weights are changed according to the performance of the decision rule. Misclassified features are given a higher weight, and the next decision rule is boosted on the reweighted training set. In this way, a series of training sets and decision rules are obtained, which are then combined in a final decision rule by simple or weighted majority voting. See, for example, "Experiments with a new boosting algorithm," Proceedings 13th International Conference on Machine Learning, 1996, pp. 148-156, the entire contents of which are incorporated herein by reference.

[0178] The measurement data used in the methods, systems, kits and compositions disclosed herein may be normalized.Normalization refers to the process of correcting, for example, the difference in the amount of assayed gene or protein level and the variability of the quality of the template used to remove the unwanted source of systematic variation measurement involved in the processing and detection of gene or protein expression.Another source of systematic variation may be due to the processing conditions in the laboratory.

[0179] In some cases, normalization methods are used to normalize laboratory processing conditions.Non-limiting examples of laboratory processing normalization that can be used in the methods of the present disclosure include but are not limited to: taking into account the systematic differences between the instruments, reagents and equipment used during the data generation process, and / or between data and the time or time course of data collection.

[0180] The assay can provide normalization by incorporating the expression of a certain normalization standard gene or protein that is known to have no significant difference in expression level under relevant conditions, i.e., a stable and constant expression level in that particular sample type. Suitable normalization genes and proteins that can be used in the present disclosure include housekeeping genes (see, for example, E. Eisenberg et al., Trends in Genetics 19(7):362-365(2003)). In some applications, normalization biomarkers (genes and proteins), also referred to as reference genes, are known to not show significantly different expression levels in subjects with TTR-CM compared to control subjects without TTR-CM. In some applications, it may be useful to add stable isotope-labeled standards that can be used for data normalization and that correspond to entities with known properties for use in data normalization.

[0181] In other applications, standard immobilized samples can be measured with every analytical batch to account for instrument and day-to-day measurement variability.

[0182] Machine learning algorithms for subselecting discriminatory biomarkers and possibly target features, and for constructing classification models are used in some methods and systems herein to determine clinical outcome scores.Examples of such algorithms are described above.These algorithms help select important biomarker features and can convert the underlying measurements into scores or probabilities for, for example, clinical outcomes, disease risk, disease likelihood, disease presence or absence, treatment response and / or disease state classification.

[0183] The ATTR biomarker score can be determined by comparing the subject-specific ATTR biomarker profile to a reference ATTR biomarker profile. The reference ATTR biomarker profile can represent a known diagnosis. For example, the ATTR biomarker profile can represent a positive diagnosis of TTR-CM. As another example, the reference ATTR biomarker profile can represent a negative diagnosis of TTR-CM. In some cases, an increase in the score indicates a higher likelihood of one or more of the following: poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and treatment recommendation for disease management. In some cases, a decrease in the quantitative score indicates a higher likelihood of one or more of the following: poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and treatment recommendation for disease management.

[0184] An ATTR biomarker profile from the control that is similar to the reference ATTR biomarker profile often indicates a higher likelihood of one or more of the following: poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and treatment recommendation for disease management. In some applications, an ATTR biomarker profile that is dissimilar between the subject and the reference indicates a higher likelihood of one or more of the following: poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and treatment recommendation for disease management.

[0185] The results can be provided to the subject, a health care professional or other professional, optionally accompanied by health recommendations, such as, for example, a recommendation to confirm or independently assess the risk of TTR-CM using one or more cardiomyopathy tests.

[0186] The recommendation may include information related to treatment regimen, such as information indicating treatment regimen.The effectiveness of the regimen may be evaluated by comparing the ATTR biomarker profile of the subject at an early time point, optionally before treatment, and at a later second time point, optionally after treatment.The ATTR biomarker profile may be compared with each other, with a reference, or otherwise evaluated to determine whether the treatment regimen shows efficacy and therefore should be continued, increased, replaced with an alternative regimen, or discontinued because it has been successful in dealing with TTR-CM or related signs and symptoms.Some evaluations rely on the comparison of the ATTR biomarker profile of the subject at multiple time points, such as at least one time point before treatment and at least one time point after treatment.The ATTR biomarker profile may be compared with each other and at least one reference biomarker panel level, or may be compared with each other and at least one reference biomarker panel level.

[0187] Treatment of amyloid transthyretin cardiomyopathy Therapeutic approaches to ATTR amyloidosis include reducing the production of TTR, inhibiting or reducing the aggregation of TTR, inhibiting or reducing TTR fibril or amyloid formation, reducing or removing TTR deposits, and stabilizing the non-toxic conformation of TTR (e.g., tetrameric form). In some embodiments, the method disclosed herein further comprises detecting the respective levels of one or more ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof) in a sample obtained from the subject, and administering an effective amount of a transthyretin (TTR) stabilizer to the diagnosed subject. In some embodiments, the TTR stabilizer is tafamidis.

[0188] In some embodiments, the disclosure includes a method of selecting a patient for treatment with a TTR stabilizer, comprising detecting the level of each of one or more ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof) in a sample obtained from the subject.

[0189] In some embodiments, the present disclosure includes a method of treating TTR-CM in a subject at risk for or suffering from TTR-CM, comprising administering a therapeutically effective amount of a TTR stabilizer to the subject, wherein the subject expresses a level of an ATTR biomarker or an ATTR biomarker gene product above a threshold value. In some embodiments, a method of treating TTR-CM in a subject at risk for or suffering from TTR-CM includes administering a therapeutically effective amount of a TTR stabilizer to the subject, wherein the subject expresses a level of more than one ATTR biomarker or an ATTR biomarker gene product above a threshold value. In some embodiments, the method further includes determining that the subject expresses a level of an ATTR biomarker or an ATTR biomarker gene product above a threshold value. In some embodiments, prior to administration, it is determined that the subject expresses a level of an ATTR biomarker or an ATTR biomarker gene product above a threshold value.

[0190] In some embodiments, the biomarkers disclosed herein can be used to screen patients for effective therapy of ATTR amyloidosis and TTR-CM. In some embodiments, the therapy of TTR-CM is a stabilizer of TTR tetramer. Examples of TTR stabilizers include tafamidis and diflunisal.

[0191] Another approach to therapy is to reduce total TTR production. For example, antisense oligonucleotide (ASO)-based therapy and RNA interference (RNAi) are therapeutic approaches to lower total TTR. ISIS-TTR Rx is an ASO-based therapy that causes the destruction of both wild-type and mutant TTR transcripts. Other possible therapeutic agents include ALN-TTR02 (patisiran), ALN-TTRsc (revusiran), doxycycline, tauroursodeoxycholic acid (TUDCA), a combination of doxycycline and TUDCA, epigallocatechin gallate (EGCG), curcumin or resveratrol. Antibodies that target TTR can also be used as therapeutic agents, for example, by antibody-mediated inhibition of TTR aggregation and fibril formation; antibody-mediated stabilization of non-toxic conformation of TTR (e.g., tetrameric form); or antibody-mediated clearance of aggregated TTR, oligomeric TTR or monomeric TTR. In addition, antibodies against TTR can be bound or conjugated to therapeutic agents, for example, to target TTR.

[0192] kit Kits comprising one or more anti-ATTR biomarker drugs and instructions for use (e.g., therapeutic, prophylactic or diagnostic use) are also provided by the present disclosure. In some embodiments, the kits are used in in vitro diagnostic assays for diagnosing TTR-CM (including TTR-CM caused by wild-type ATTR amyloidosis). In some embodiments, the kits of the present disclosure further comprise a TTR stabilizer (e.g., tafamidis).

[0193] In some embodiments, the one or more anti-ATTR biomarker drugs comprise an antibody drug. In some embodiments, one or more of the antibody drugs are labeled with a detectable moiety. In some embodiments, the kit further comprises a detection agent (e.g., one or more acridinium ester molecules). In some embodiments, one or more of the antibody drugs are labeled with one or more acridinium ester molecules. In some embodiments, the kit further comprises one or more secondary antibody drugs that specifically bind to one or more of the anti-ATTR biomarker antibody drugs.

[0194] In some embodiments, one or more anti-ATTR biomarker drugs comprise a nucleic acid probe. In some embodiments, at least a portion of each nucleic acid probe hybridizes to one or more portions of a nucleotide that encodes an ATTR biomarker (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof). The nucleotide that encodes the ATTR biomarker may be DNA (e.g., cDNA) or RNA (e.g., mRNA). In some embodiments, the nucleic acid probe is labeled with one or more detection agents (e.g., the detection agent indicates the presence of the nucleotide that encodes the ATTR biomarker).

[0195] In some embodiments, the kit further comprises one or more control samples. In some embodiments, the control samples comprise one or more ATTR biomarker standards. In some embodiments, the ATTR biomarker standards comprise recombinant ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or combinations thereof). In some embodiments, the ATTR biomarker standards comprise synthetic ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or combinations thereof) nucleic acids.

[0196] In addition to the above, the kit can include other components, such as solvents or buffers, stabilizers or preservatives, and / or agents for treating a condition or disorder described herein. Alternatively, the other components can be included in the kit, but in a different composition or container than the anti-ATTR biomarker drug. In such embodiments, the kit can include instructions for mixing the anti-ATTR biomarker drug with the other components, or for using the anti-ATTR biomarker with the other components.

[0197] In certain embodiments, kits for use in accordance with the present disclosure can include reference or control samples, instructions for processing the samples and performing tests on the samples, instructions for interpreting the results, buffers and / or other reagents necessary for performing the tests.

[0198] The present disclosure also provides the recognition that a certain single ATTR biomarker can be useful for detecting and / or diagnosing ATTR amyloidosis or TTR-CM.The present disclosure further provides the insight that a certain combination of ATTR biomarkers is particularly useful for detecting and / or diagnosing ATTR amyloidosis or TTR-CM.Therefore, the methods, compositions and kits described herein can be used in assays to assess the risk of TTR-CM, to assess whether a subject should undergo further cardiac testing, and / or to diagnose TTR-CM based on the detection or measurement of ATTR biomarkers in a sample, for example, a biological sample obtained from a subject.

[0199] The methods and kits provided herein can detect TTR-CM in a sample with a sensitivity and specificity that makes the results of the test medically practicable and reliable. The methods and kits provided herein for detecting and / or diagnosing TTR-CM in a subject can detect TTR-CM with a sensitivity of greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or about 100%. In some embodiments, the methods and kits provided herein can detect TTR-CM with a sensitivity of between about 70%-100%, between about 80%-100%, or between about 90-100%. In some embodiments, the methods and kits provided herein can detect TTR-CM with a specificity of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or about 100%. In some embodiments, the methods and kits provided herein can detect TTR-CM with a specificity of between about 50%-100%, between about 60%-100%, between about 70%-100%, between about 80%-100%, or between about 90-100%. In some embodiments, the methods and kits provided herein can detect TTR-CM with a sensitivity and specificity of 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more. In some embodiments, the methods and kits provided herein can detect TTR-CM with a sensitivity and specificity of between about 50%-100%, between about 60%-100%, between about 70%-100%, between about 80%-100%, or between about 90-100%.

[0200] composition Compositions are also provided herein. In some embodiments, the composition comprises one or more ATTR biomarkers and one or more anti-ATTR biomarker drugs. In some embodiments, the one or more ATTR biomarkers comprise TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL or a combination thereof, and the one or more anti-ATTR biomarker drugs comprise anti-TnI drugs, anti-PKM1 drugs, anti-PKM2 drugs, anti-NT-proBNP drugs, anti-RBP4 drugs, anti-TIMP2 drugs, anti-NfL drugs or a combination thereof.

[0201] In some embodiments, the compositions include a combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, five or more, etc.) and a corresponding combination of anti-ATTR biomarker drugs.

[0202] In some embodiments, the composition comprises two or more ATTR biomarkers and two or more anti-ATTR biomarker drugs. In some embodiments, the two or more ATTR biomarkers comprise TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof, and the two or more anti-ATTR biomarker drugs comprise an anti-TnI drug, an anti-PKM1 drug, an anti-PKM2 drug, an anti-NT-proBNP drug, an anti-RBP4 drug, an anti-TIMP2 drug, an anti-NfL drug, or a combination thereof.

[0203] In some embodiments, the composition comprises three or more ATTR biomarkers and three or more anti-ATTR biomarker drugs.In some embodiments, the three or more ATTR biomarkers comprise TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof.In some embodiments, the three or more anti-ATTR biomarker drugs comprise anti-TnI drugs, anti-PKM1 drugs, anti-PKM2 drugs, anti-NT-proBNP drugs, anti-RBP4 drugs, anti-TIMP2 drugs, anti-NfL drugs, or a combination thereof.

[0204] In some embodiments, the composition comprises four or more ATTR biomarkers and four or more anti-ATTR biomarker drugs. In some embodiments, the four or more ATTR biomarkers comprise TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof. In some embodiments, the four or more anti-ATTR biomarker drugs comprise anti-TnI drugs, anti-PKM1 drugs, anti-PKM2 drugs, anti-NT-proBNP drugs, anti-RBP4 drugs, anti-TIMP2 drugs, anti-NfL drugs, or a combination thereof.

[0205] In some embodiments, the composition comprises five or more ATTR biomarkers and five or more anti-ATTR biomarker drugs.In some embodiments, the five or more ATTR biomarkers comprise TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof.In some embodiments, the five or more anti-ATTR biomarker drugs comprise anti-TnI drugs, anti-PKM1 drugs, anti-PKM2 drugs, anti-NT-proBNP drugs, anti-RBP4 drugs, anti-TIMP2 drugs, anti-NfL drugs, or a combination thereof.

[0206] In some embodiments, the composition comprises a combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) and a corresponding combination of anti-ATTR biomarker drugs. In some embodiments, the composition comprises a combination of ATTR biomarkers and a corresponding combination of anti-ATTR biomarker drugs in Tables 2-5. In some embodiments, the composition comprises a combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) and a corresponding combination of anti-ATTR biomarker drugs in Table 2. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) and a corresponding combination of anti-ATTR biomarker drugs in Table 3. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) and a corresponding combination of anti-ATTR biomarker drugs in Table 4. In some embodiments, a combination of the ATTR biomarkers of Table 5 (eg, one or more, two or more, three or more, four or more, etc.) and a corresponding combination of anti-ATTR biomarker drugs.

[0207] In some embodiments, the composition comprises TnI and an anti-TnI drug. In some embodiments, the composition comprises TnI, PKM1, an anti-TnI drug and an anti-PKM1 drug. In some embodiments, the composition comprises TnI, PKM2, an anti-TnI drug and an anti-PKM2 drug. In some embodiments, the composition comprises TnI, PKM1, PKM2, an anti-TnI drug, an anti-PKM1 drug and an anti-PKM2 drug. In some embodiments, the composition comprises NT-proBNP, RBP4 or both and an anti-NT-proBNP drug, an anti-RBP4 drug or both. In some embodiments, the composition comprises TIMP2, NfL or both and an anti-TIMP2 drug, an anti-NfL drug or both.

[0208] In some embodiments, the composition comprises NT-proBNP and an anti-NT-proBNP drug. In some embodiments, the composition comprises TnI, PKM1, PKM2, RBP4 or a combination thereof and an anti-TnI drug, an anti-PKM1 drug, an anti-PKM2 drug, an anti-RBP4 drug or a combination thereof. In some embodiments, the composition comprises TIMP2, NfL or both and an anti-TIMP2 drug, an anti-NfL drug or both.

[0209] In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes TnI, PKM2, NT-proBNP, RBP4, anti-TnI drugs, anti-PKM2 drugs, anti-NT-proBNP drugs, and anti-RBP4 drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes TnI, PKM2, NT-proBNP, anti-TnI drugs, anti-NT-proBNP drugs, and anti-RBP4 drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes TnI, PKM2, RBP4, anti-TnI drugs, anti-PKM2 drugs, and anti-RBP4 drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes TnI, PKM1, NT-proBNP, anti-TnI drugs, anti-PKM1 drugs, and anti-NT-proBNP drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes TnI, PKM1, RBP4, anti-TnI drugs, anti-PKM1 drugs, and anti-RBP4 drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes TnI, PKM1, PKM2, NT-proBNP, RBP4, anti-TnI drugs, anti-PKM1 drugs, anti-PKM2 drugs, anti-NT-proBNP drugs, and anti-RBP4 drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes TnI, PKM1, PKM2, NT-proBNP, an anti-TnI drug, an anti-PKM1 drug, an anti-PKM2 drug, and an anti-NT-proBNP drug. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes TnI, PKM1, PKM2, RBP4, an anti-TnI drug, an anti-PKM1 drug, an anti-PKM2 drug, and an anti-RBP4 drug.In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes NT-proBNP, RBP4, TnI, anti-TnI drugs, anti-NT-proBNP drugs, and anti-RBP4 drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes RBP4, SMOC-2, TnI, anti-TnI drugs, anti-RBP4 drugs, and anti-SMOC-2 drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes DCN, NT-proBNP, TnI, anti-TnI drugs, anti-NT-proBNP drugs, and anti-DCN drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes DCN, RBP4, TnI, anti-TnI drugs, anti-RBP4 drugs, and anti-DCN drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes NT-proBNP, SMOC-2, TnI, anti-TnI drugs, anti-NT-proBNP drugs, and anti-SMOC-2 drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes NT-proBNP, TIMP2, TnI, anti-TnI drugs, anti-NT-proBNP drugs, and anti-TIMP2 drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes NT-proBNP, TnI, anti-TnI drugs, and anti-NT-proBNP drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes DCN, TnI, anti-TnI drugs, and anti-DCN drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes DCN, TIMP2, TnI, anti-TnI drugs, anti-TIMP2 drugs, and anti-DCN drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes RBP4, TnI, anti-TnI drugs, and anti-RBP4 drugs.In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes RBP4, TIMP2, TnI, anti-TnI drugs, anti-RBP4 drugs, and anti-TIMP2 drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes DCN, SMOC-2, TnI, anti-TnI drugs, anti-SMOC-2 drugs, and anti-DCN drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes TIMP2, TnI, anti-TnI drugs, and anti-TIMP2 drugs. In some embodiments, the combination of ATTR biomarkers (e.g., one or more, two or more, three or more, four or more, etc.) includes SMOC-2, TIMP2, TnI, anti-TnI drugs, anti-SMOC-2 drugs, and anti-TIMP2 drugs.

[0210] In some embodiments, one or more of the anti-ATTR biomarker drugs in the compositions provided herein comprise an antibody drug. In some embodiments, one or more of the antibody drugs are labeled with a detectable moiety. In some embodiments, the kit further comprises a detection agent (e.g., one or more acridinium ester molecules). In some embodiments, one or more of the antibody drugs are labeled with one or more acridinium ester molecules. In some embodiments, the kit further comprises one or more secondary antibody drugs that specifically bind to one or more of the anti-ATTR biomarker antibody drugs.

[0211] In some embodiments, one or more anti-ATTR biomarker drugs in the compositions provided herein comprise a nucleic acid probe. In some embodiments, at least a portion of each nucleic acid probe hybridizes to one or more portions of the nucleotides encoding an ATTR biomarker (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof). The nucleotides encoding the ATTR biomarkers may be DNA (e.g., cDNA) or RNA (e.g., mRNA). In some embodiments, the nucleic acid probe is labeled with one or more detection agents (e.g., the detection agent indicates the presence of the nucleotides encoding the ATTR biomarker).

[0212] In some embodiments, the composition comprises one or more control samples. In some embodiments, the control samples comprise one or more ATTR biomarker standards. In some embodiments, the ATTR biomarker standards comprise recombinant ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof). In some embodiments, the ATTR biomarker standards comprise synthetic ATTR biomarkers (e.g., TnI, PKM1, PKM2, NT-proBNP, RBP4, DCN, TIMP2, SMOC-2, NfL, or a combination thereof) nucleic acids.

[0213] In addition to the above, the compositions can include other components, such as solvents or buffers, stabilizers or preservatives, and / or agents for treating a condition or disorder described herein.

[0214] Computer Systems The methods described herein can be implemented in a computer system having a processor that executes certain instructions in a computer program.In some embodiments, the computer system can be configured to output a score of ATTR biomarkers based on receiving an ATTR biomarker profile and / or the level of two or more ATTR biomarkers.In particular, the computer program can include instructions for the system to select an appropriate next step, such as further medication (e.g., TTR stabilizer), treatment, and / or further test (e.g., cardiomyopathy test) for the subject.

[0215] In some embodiments, the computer program can be configured to allow the computer system to identify subjects for further testing (e.g., cardiomyopathy testing), identify subjects as at risk for or having TTR-CM, and / or identify patients for receiving medication (e.g., TTR stabilizers) based on the received data (e.g., ATTR biomarker profile), and calculate the ATTR biomarker score using the data. The system may be able to rank the identified next steps based on the ATTR biomarker profile together with demographic factors and / or image-based biomarkers. The system can adjust the ranking based, for example, on the clinical response of the subject, or of the subject having or suspected of having TTR-CM, or of the subject's family.

[0216] 11 is a block diagram of a computer system 1100 that can be used in the above operations, according to one embodiment. The system 1100 includes a processor 1110, a memory 1120, a storage device 1130, and an input / output device 1140. Each of the components 1110, 1120, 1130, and 1140 are interconnected using a system bus 1150. The system can include an analytical instrument 1160 for determining the level of one or more ATTR biomarkers in a sample.

[0217] The processor 1110 may process instructions for execution within the system 1100. In one embodiment, the processor 1110 is a single-threaded processor. In another embodiment, the processor 1110 is a multi-threaded processor. The processor 1110 may process instructions stored in the memory 1120 or the storage device 1130, including for receiving or transmitting information through the input / output device 1140.

[0218] The memory 1120 stores information within the system 1100. In one embodiment, the memory 1120 is a computer-readable medium. In one embodiment, the memory 1120 is a volatile memory unit. In another embodiment, the memory 1120 is a non-volatile memory unit.

[0219] The storage device 1130 is capable of providing mass storage for the system 1100. In one embodiment, the storage device 1130 is a computer-readable medium.

[0220] The input / output device(s) 1140 provide input / output operations to the system 1100. In one embodiment, the input / output device(s) 1140 include a keyboard and / or a pointing device. In one embodiment, the input / output device(s) 1140 include a display unit for displaying a graphical user interface.

[0221] System 1100 can be used to build a database. Figure 12 shows a flow chart of a method 1200 for building a database for use in identifying subjects for further testing (e.g., cardiomyopathy testing), identifying subjects as at risk for or having TTR-CM, and / or identifying patients for receiving medication (e.g., TTR stabilizers). Preferably, method 1200 is implemented in system 1100. For example, a computer program product can include instructions to cause processor 1110 to perform the steps of method 1200 or method 1300.

[0222] Method 1200 includes the following steps: In step 1210, a subject's ATTR biomarker profile (e.g., levels of one or more ATTR biomarkers in a sample) is received. The computer program in system 600 can include instructions for presenting a suitable graphical user interface on input / output device 640, which can prompt a user to input a level 670 using input / output device 640, e.g., a keyboard. In step 1220, an ATTR biomarker score is calculated from the ATTR biomarker profile. As described herein, in step 1220, an ATTR biomarker score is calculated from (i) the ATTR biomarker profile and (ii) demographic factors and / or image-based biomarkers. In step 1230, the ATTR biomarker score is stored. System 600 can store the ATTR biomarker score in storage device 630. Additionally or alternatively, system 600 can provide a readout including the ATTR biomarker score. The readout can also include a confidence level associated with the next step proposed for the subject and / or the ATTR biomarker score.

[0223] Method 1300 includes the following steps: In step 1310, detect the level of one or more ATTR biomarkers, for example in a sample from a subject. In step 1320, obtain an ATTR biomarker profile using the level of one or more ATTR biomarkers. In step 1330, calculate an ATTR biomarker score from the ATTR biomarker profile. In step 1330, calculate an ATTR biomarker score from (i) the ATTR biomarker profile and (ii) demographic factors and / or image-based biomarkers, as described herein. In step 1340, store the ATTR biomarker score. System 600 can store the ATTR biomarker score in storage device 630. Additionally or alternatively, system 600 can provide a readout including the ATTR biomarker score. The readout can also include a confidence level associated with the next step proposed for the subject and / or the ATTR biomarker score.

[0224] Further provided is a non-transitory computer readable medium that includes executable instructions that, when executed, cause a processor to perform operations including the methods provided herein. For example, a non-transitory computer readable medium that includes executable instructions that, when executed, cause a processor to perform operations including the methods of 1200 or 1300 above.

[0225] Numbered Exemplary Embodiments Embodiment 1. A method, the method comprising detecting a respective level of two or more transthyretin amyloidosis (ATTR) biomarkers in a sample, the two or more ATTR biomarkers comprising: (i) troponin I (TnI); (ii) pyruvate kinase muscle isoform 1 (PKM1); (iii) pyruvate kinase muscle isoform 2 (PKM2); (iv) N-terminal-prohormone B-type natriuretic peptide (NT-proBNP); (v) retinol binding protein 4 (RBP4); (vi) tissue inhibitor of metalloproteinase 2 (TIMP2); (vii) neurofilament light chain (NfL); or (viii) a combination thereof.

[0226] Embodiment 2. The method of embodiment 1, wherein the level of each of three or more ATTR biomarkers in the sample is detected, the three or more ATTR biomarkers comprising: (i) TnI, (ii) PKM1, (iii) PKM2, (iv) NT-proBNP, (v) RBP4, (vi) TIMP2, (vii) NfL, or (viii) a combination thereof.

[0227] Embodiment 3. The method of embodiment 1 or 2, wherein the levels of four or more ATTR biomarkers in the sample are detected, the four or more ATTR biomarkers comprising: (i) TnI, (ii) PKM1, (iii) PKM2, (iv) NT-proBNP, (v) RBP4, (vi) TIMP2, (vii) NfL, or (viii) a combination thereof.

[0228] Embodiment 4. A method of detecting levels of two or more ATTR biomarkers in a sample, wherein the two or more ATTR biomarkers include TnI.

[0229] Embodiment 5. The method of embodiment 4, wherein the two or more ATTR biomarkers include TnI and PKM1.

[0230] Embodiment 6 The method of embodiment 4, wherein the two or more ATTR biomarkers include TnI and PKM2.

[0231] Embodiment 7 The method of embodiment 4, wherein the two or more ATTR biomarkers include TnI, PKM1 and PKM2.

[0232] Embodiment 8. The method of any one of embodiments 4-7, wherein the two or more ATTR biomarkers further comprise NT-proBNP, RBP4, or both.

[0233] Embodiment 9. The method of any one of embodiments 4-8, wherein the two or more ATTR biomarkers further comprise TIMP2, NfL, or both.

[0234] Embodiment 10. A method of detecting the level of two or more ATTR biomarkers in a sample, wherein the two or more ATTR biomarkers comprise NT-proBNP.

[0235] Embodiment 11. The method of any one of embodiments 4-7, wherein the two or more ATTR biomarkers further comprise TnI, PKM1, PKM2, RBP4, or a combination thereof.

[0236] Embodiment 12. The method of any one of embodiments 4-8, wherein the two or more ATTR biomarkers further comprise TIMP2, NfL, or both.

[0237] Embodiment 13. A method comprising: (a) detecting the levels of two or more ATTR biomarkers in a sample to obtain an ATTR biomarker profile; and (b) computing an ATTR biomarker score using the ATTR biomarker profile. A method comprising:

[0238] Embodiment 14. The method of embodiment 13, wherein the two or more ATTR biomarkers comprise: (i) TnI, (ii) PKM1, (iii) PKM2, (iv) NT-proBNP, (v) RBP4, (vi) TIMP2, (vii) NfL, or (viii) a combination thereof.

[0239] Embodiment 15. The method of embodiment 13 or 14, wherein the levels of three or more ATTR biomarkers in the sample are detected, the three or more ATTR biomarkers comprising: (i) TnI, (ii) PKM1, (iii) PKM2, (iv) NT-proBNP, (v) RBP4, (vi) TIMP2, (vii) NfL, or (viii) a combination thereof.

[0240] Embodiment 16. The method of any one of embodiments 13-15, wherein the levels of four or more ATTR biomarkers in the sample are detected, the four or more ATTR biomarkers comprising: (i) TnI, (ii) PKM1, (iii) PKM2, (iv) NT-proBNP, (v) RBP4, (vi) TIMP2, (vii) NfL, or (viii) a combination thereof.

[0241] Embodiment 17 The method of any one of embodiments 13-16, wherein the two or more ATTR biomarkers include TnI.

[0242] Embodiment 18. The method of any one of embodiments 13-17, wherein the two or more ATTR biomarkers include TnI and PKM1.

[0243] Embodiment 19. The method of any one of embodiments 13-18, wherein the two or more ATTR biomarkers include TnI and PKM2.

[0244] Embodiment 20. The method of any one of embodiments 13-19, wherein the two or more ATTR biomarkers include TnI, PKM1 and PKM2.

[0245] Embodiment 21. The method of any one of embodiments 17-20, wherein the two or more ATTR biomarkers further comprise NT-proBNP, RBP4, or both.

[0246] Embodiment 22. The method of any one of embodiments 17-21, wherein the two or more ATTR biomarkers further comprise TIMP2, NfL, or both.

[0247] Embodiment 23. The method of any one of embodiments 13-16, wherein the two or more ATTR biomarkers include NT-proBNP.

[0248] Embodiment 24 The method of embodiment 23, wherein the two or more ATTR biomarkers further comprise TnI, PKM1, PKM2, RBP4, or a combination thereof.

[0249] Embodiment 25 The method of embodiment 23 or 24, wherein the two or more ATTR biomarkers further comprise TIMP2, NfL, or both.

[0250] Embodiment 26 The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI, PKM1, NT-proBNP and RBP4.

[0251] Embodiment 27 The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI, PKM1 and RBP4.

[0252] Embodiment 28. The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI, PKM1 and NT-proBNP.

[0253] Embodiment 29. The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI, PKM2, NT-proBNP and RBP4.

[0254] Embodiment 30 The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI, PKM2 and RBP4.

[0255] Embodiment 31. The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI, PKM2 and NT-proBNP.

[0256] Embodiment 32. The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI, PKM1, PKM2, NT-proBNP, and RBP4.

[0257] Embodiment 33. The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI, PKM1, PKM2 and RBP4.

[0258] Embodiment 34 The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI, NT-proBNP and RBP4.

[0259] Embodiment 35. The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI, NT-proBNP and TIMP2.

[0260] Embodiment 36 The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI and NT-proBNP.

[0261] Embodiment 37 The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI and RBP4.

[0262] Embodiment 38 The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI, RBP4 and TIMP2.

[0263] Embodiment 39. The method of embodiment 1, wherein the two or more ATTR biomarkers comprise or consist of TnI and TIMP2.

[0264] Embodiment 40. The method of any one of embodiments 1-3, 4, 6, 8, 9, 10-12, 13-17, 19, and 21-25, wherein the two or more ATTR biomarkers do not include PKM1.

[0265] Embodiment 41 The method of any one of embodiments 1-3, 4, 5, 8, 9, 10-12, 13-18, and 21-25, wherein the two or more ATTR biomarkers do not include PKM2.

[0266] Embodiment 42. The method of any one of embodiments 1-3, 4, 8, 9, 10-12, 13-17, and 21-25, wherein the two or more ATTR biomarkers do not include PKM1 or PKM2.

[0267] Embodiment 43 The method of any one of embodiments 1 to 42, wherein the two or more ATTR biomarkers do not include SMOC-2.

[0268] Embodiment 44 The method of any one of embodiments 1-42, wherein the two or more ATTR biomarkers do not include DCN.

[0269] Embodiment 45. The method of any one of embodiments 1-42, wherein the two or more ATTR biomarkers do not include SMOC-2 or DCN.

[0270] Embodiment 46. The method of any one of embodiments 1 to 45, wherein the method is a method of determining a subject's risk of developing amyloid transthyretin cardiomyopathy (TTR-CM), and the sample is obtained from the subject.

[0271] Embodiment 47. The method of any one of embodiments 1 to 45, wherein the method is for diagnosing a subject with TTR-CM, and the sample is obtained from the subject.

[0272] Embodiment 48 The method of any one of embodiments 1-45, which is a method of treating TTR-CM in a subject at risk for or suffering from TTR-CM.

[0273] Embodiment 49 The method of any one of embodiments 46-48, wherein the TTR-CM results from wild-type transthyretin amyloidosis (ATTRwt).

[0274] Embodiment 50. The method of any one of embodiments 46-49, wherein the subject tests negative for familial amyloidotic cardiomyopathy (ATTRm) by genetic testing.

[0275] Embodiment 51. The method of any one of embodiments 1 to 45, wherein the method is for selecting a subject to receive one or more doses of a TTR stabilizer, and the sample is obtained from the subject.

[0276] Embodiment 52 The method of embodiment 51, further comprising administering one or more doses of a TTR stabilizer to the subject.

[0277] Embodiment 53. The method of any one of embodiments 1 to 45, wherein the method is a method of selecting a subject for one or more cardiomyopathy tests, and the sample is obtained from the subject.

[0278] Embodiment 54. The method of embodiment 53, wherein the one or more cardiomyopathy tests include an echocardiogram, advanced imaging methods, or both.

[0279] Embodiment 55. The method of embodiment 54, wherein the advanced imaging method comprises cardiac magnetic resonance imaging (CMR), scintigraphy, or both.

[0280] 56. Scintigraphy is 977 56. The method of embodiment 55, comprising the use of a radioisotope conjugate, such as Tc-pyrophosphate.

[0281] Embodiment 57. The method of embodiment 55 or 56, wherein the scintigraphy is performed using single photon emission computed tomography (SPECT).

[0282] Embodiment 58 The method of any one of embodiments 1 to 45, which is a method of determining whether a patient does not have TTR-CM or is not at risk of developing TTR-CM.

[0283] Embodiment 59. The method of any one of embodiments 1 to 57, wherein the sample is or comprises a biological sample.

[0284] Embodiment 60. The method of embodiment 59, wherein the biological sample comprises blood, serum, plasma or cardiac tissue.

[0285] Embodiment 61. The method of any one of embodiments 1 to 60, wherein the sample is obtained from a subject.

[0286] Embodiment 62 The method of embodiment 61, wherein the subject is a human.

[0287] Embodiment 63 The method of embodiment 61 or 62, wherein the subject has or is at risk of developing amyloid transthyretin cardiomyopathy (TTR-CM).

[0288] Embodiment 64. The method of any one of embodiments 46-63, wherein the subject is at least 65 years of age.

[0289] Embodiment 65. The method of any one of embodiments 1 to 64, wherein detecting the levels of two or more ATTR biomarkers in the sample comprises detecting the presence, absence, level or genotype of each of the two or more ATTR biomarkers in the sample.

[0290] Embodiment 66. The method of any one of embodiments 1 to 65, wherein detecting the levels of two or more ATTR biomarkers in the sample comprises performing mass spectrometry.

[0291] Embodiment 67. The method of any one of embodiments 1 to 66, wherein detecting the levels of two or more ATTR biomarkers in the sample comprises measuring chemiluminescence.

[0292] Embodiment 68. The method of any one of embodiments 1 to 67, wherein detecting the levels of two or more ATTR biomarkers in the sample comprises detecting two or more nucleotides encoding the two or more ATTR biomarkers in the sample.

[0293] Embodiment 69. The method of embodiment 68, wherein detecting two or more nucleotides encoding two or more ATTR biomarkers comprises performing a nucleic acid amplification method.

[0294] Embodiment 70. The method of embodiment 69, wherein the nucleic acid amplification method is selected from the group consisting of polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-specific amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA).

[0295] Embodiment 71. The method of any one of embodiments 68 to 70, wherein detecting two or more nucleotides encoding two or more ATTR biomarkers comprises detecting hybridization between two or more nucleic acid probes and two or more nucleotides encoding two or more ATTR biomarkers.

[0296] Embodiment 72. The method of embodiment 71, wherein the two or more nucleic acid probes are each complementary to at least a portion of one of the two or more nucleotides encoding the two or more ATTR biomarkers.

[0297] Embodiment 73 The method of any one of embodiments 68-72, wherein the nucleotide sequence encoding two or more ATTR biomarkers is DNA.

[0298] Embodiment 74 The method of embodiment 73, wherein the DNA is cDNA.

[0299] Embodiment 75 The method of any one of embodiments 68-72, wherein the nucleotide sequence encoding the two or more ATTR biomarkers is RNA.

[0300] Embodiment 76. The method of any one of embodiments 1 to 75, wherein detecting the levels of two or more ATTR biomarkers in the sample comprises performing an immunoassay.

[0301] Embodiment 77 The method of embodiment 76, wherein the immunoassay is a chemiluminescence immunoassay.

[0302] Embodiment 78. The method of embodiment 76 or 77, wherein the immunoassay is selected from the group consisting of: immunoprecipitation; Western blot; ELISA; immunohistochemistry; immunocytochemistry; flow cytometry; and immuno-PCR.

[0303] Embodiment 79. The method of any one of embodiments 76 to 78, wherein the immunoassay is an ELISA.

[0304] Embodiment 80. The method of any one of embodiments 76 to 79, wherein the immunoassay is a high-throughput and / or automated immunoassay platform.

[0305] Embodiment 81. The method of any one of embodiments 1 to 80, wherein detecting the levels of two or more ATTR biomarkers in the sample comprises contacting the sample with two or more anti-ATTR biomarker antibody drugs.

[0306] Embodiment 82. The method of any one of embodiments 1 to 81, wherein detecting the levels of two or more ATTR biomarkers in a sample obtained from the subject comprises contacting the sample with two or more detection agents.

[0307] Embodiment 83 The method of embodiment 82, wherein the two or more anti-ATTR biomarker antibody drugs are labeled with two or more detection agents.

[0308] Embodiment 84 The method of embodiment 82 or 83, wherein the two or more detection agents are two or more acridinium ester molecules.

[0309] Embodiment 85. The method of any one of embodiments 1 to 84, wherein detecting the levels of two or more ATTR biomarkers in the sample comprises detecting binding between the two or more ATTR biomarkers and two or more anti-ATTR biomarker antibody drugs.

[0310] Embodiment 86 The method of embodiment 85, wherein detecting binding between two or more ATTR biomarkers and two or more anti-ATTR biomarker antibody drugs comprises performing immunocytochemistry (ICC).

[0311] Embodiment 87. The method of embodiment 85 or 86, wherein detecting binding between the two or more ATTR biomarkers and the two or more anti-ATTR biomarker antibody drugs comprises determining an absorbance or luminescence value of at least one ATTR biomarker.

[0312] Embodiment 88. The method of any one of embodiments 1 to 87, wherein detecting the levels of two or more ATTR biomarkers in a sample comprises detecting the level of each of the two or more ATTR biomarkers, and further comprises comparing the level of each of the two or more ATTR biomarkers to a threshold value for each ATTR biomarker, wherein each of the detected levels of the one or more ATTR biomarkers is above the threshold value for each ATTR biomarker.

[0313] Embodiment 89 The method of embodiment 88, further comprising determining that the subject has or is at risk of developing TTR-CM if the respective levels of the one or more ATTR biomarkers are above a threshold value for the respective ATTR biomarker.

[0314] Embodiment 90 The method of embodiment 88 or 89, further comprising diagnosing the subject with TTR-CM if the respective levels of the one or more ATTR biomarkers are above a threshold value for the respective ATTR biomarker.

[0315] Embodiment 91. The method of any one of embodiments 88-90, further comprising the step of recommending the subject for one or more cardiomyopathy trials if the respective levels of the one or more ATTR biomarkers are above a threshold value for the respective ATTR biomarker.

[0316] Embodiment 92. The method of any one of embodiments 88-91, wherein the level of each of the one or more ATTR biomarkers is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, or at least 1.9 times greater than the threshold value for the respective ATTR biomarker.

[0317] Embodiment 93. The method of any one of embodiments 88-92, wherein the threshold value for the ATTR biomarker is the average of the values ​​for the ATTR biomarker detected for two or more control samples.

[0318] Embodiment 94 The method of embodiment 93, wherein each of the two or more control samples is a sample obtained from a subject not having ATTR amyloidosis.

[0319] Embodiment 95. The method of any one of embodiments 88-92, wherein the threshold value for the ATTR biomarker is a value reported in a standard table.

[0320] Embodiment 96. The method of any one of embodiments 88-92, further comprising the step of recommending the subject for a cardiac biopsy if the respective levels of the one or more ATTR biomarkers are above a threshold, and the subject tests positive for cardiomyopathy in one or more of the cardiomyopathy tests.

[0321] Embodiment 97. The method of any one of embodiments 13-87, wherein using the ATTR biomarker profile to compute an ATTR biomarker score comprises applying an algorithm to the ATTR biomarker profile to compute an ATTR biomarker score, wherein the algorithm is a decision tree algorithm, a neural boosting algorithm, a bootstrap forest algorithm, a boosted tree algorithm, a K-nearest neighbor algorithm, a generalized regression variable forward algorithm, a generalized regression pruning variable forward algorithm, a fit stepwise algorithm, a generalized regression lasso algorithm, a generalized regression elastic net algorithm, a generalized regression ridge algorithm, a nominal logistic algorithm, a support vector machine algorithm, a discriminant algorithm, or a naive Bayes algorithm.

[0322] Embodiment 98. The method of any one of embodiments 13-87 and 97, wherein the step of computing an ATTR biomarker score using the ATTR biomarker profile comprises applying an algorithm to the ATTR biomarker profile to compute an ATTR biomarker score, wherein the algorithm is a decision tree algorithm, a neural boosting algorithm, a bootstrap forest algorithm, a boosted tree algorithm, a generalized regression lasso algorithm, a generalized regression elastic net algorithm, a generalized regression ridge algorithm, a nominal logistic algorithm, a support vector machine algorithm, or a discrimination algorithm.

[0323] Embodiment 99. The method of any one of embodiments 13-87, 97 and 98, wherein the step of computing an ATTR biomarker score using the ATTR biomarker profile comprises applying an algorithm to the ATTR biomarker profile to compute an ATTR biomarker score, wherein the algorithm is a decision tree algorithm, a neural boosting algorithm, a bootstrap forest algorithm, a boosted tree algorithm or a support vector machine algorithm.

[0324] Embodiment 100. The method of any one of embodiments 13-87 and 97-99, further comprising using the score of ATTR biomarkers to determine whether the subject from which the sample was obtained is at risk for or has TTR-CM.

[0325] Embodiment 101. The method of any one of embodiments 13-87 and 97-100, further comprising using the score of the ATTR biomarker to determine whether the subject from whom the sample was obtained is selected for one or more cardiomyopathy tests.

[0326] Embodiment 102. The method of any one of embodiments 13-87 and 97-101, further comprising using the score of ATTR biomarkers to determine whether the subject from whom the sample was obtained is selected to receive one or more doses of a TTR stabilizer.

[0327] Embodiment 103. The method of any one of embodiments 1 to 102, further comprising immunochemical staining of biopsy tissue from the subject.

[0328] Embodiment 104. The method of embodiment 88, wherein the biopsy tissue comprises cardiac biopsy tissue.

[0329] Embodiment 105. The method of embodiment 88 or 89, wherein the immunochemical staining comprises the use of two or more antibody drugs against kappa or lambda light chain amyloid deposits and / or two or more antibody drugs against transthyretin deposits in cardiac tissue.

[0330] Embodiment 106 The method of any one of embodiments 88-90, further comprising diagnosing the subject with TTR-CM if immunochemical staining indicates the presence of transthyretin deposits in cardiac tissue.

[0331] Embodiment 107. (a) detecting the levels of two or more ATTR biomarkers in a sample obtained from a subject to obtain an ATTR biomarker profile; and (b) computing an ATTR biomarker score using the ATTR biomarker profile. A method comprising: the two or more ATTR biomarkers include (i) TnI, (ii) PKM1, (iii) PKM2, (iv) NT-proBNP, (v) RBP4, (vi) TIMP2, (vii) NfL, or (viii) a combination thereof; The method, wherein computing an ATTR biomarker score using the ATTR biomarker profile comprises applying an algorithm to the ATTR biomarker profile to compute an ATTR biomarker score, wherein the algorithm is a decision tree algorithm, a neural boosting algorithm, a bootstrap forest algorithm, a boosted tree algorithm, or a support vector machine algorithm.

[0332] Embodiment 108. The method of embodiment 107, wherein the subject is a human subject.

[0333] Embodiment 109. The method of embodiment 107 or 108, wherein the subject is at least 65 years of age.

[0334] Embodiment 110. The method of any one of embodiments 107 to 109, wherein the sample comprises blood, serum, plasma or cardiac tissue.

[0335] Embodiment 111. The method of any one of embodiments 107-110, further comprising using the score of ATTR biomarkers to determine whether the subject from which the sample was obtained is at risk for or has TTR-CM.

[0336] Embodiment 112. The method of any one of embodiments 107 to 111, further comprising using the score of ATTR biomarkers to determine the subject's risk of developing amyloid transthyretin cardiomyopathy (TTR-CM).

[0337] Embodiment 113. The method of any one of embodiments 107-112, further comprising diagnosing the subject with TTR-CM using the score of ATTR biomarkers.

[0338] Embodiment 114 The method of embodiment 113, wherein the TTR-CM results from wild-type transthyretin amyloidosis (ATTRwt).

[0339] Embodiment 115. The method of any one of embodiments 107 to 114, further comprising using the score of the ATTR biomarker to determine whether the subject from whom the sample was obtained is selected for one or more cardiomyopathy trials.

[0340] Embodiment 116. The method of embodiment 115, wherein the one or more cardiomyopathy tests include an echocardiogram, advanced imaging methods, or both.

[0341] Embodiment 117. The method of embodiment 116, wherein the advanced imaging method includes cardiac magnetic resonance imaging (CMR), scintigraphy, or both.

[0342] Embodiment 118. Scintigraphy is 99m 118. The method of embodiment 117, comprising the use of a radioisotope conjugate, such as Tc-pyrophosphate.

[0343] Embodiment 119. The method of embodiment 117 or 118, wherein the scintigraphy is performed using single photon emission computed tomography (SPECT).

[0344] Embodiment 120. The method of any one of embodiments 107 to 119, further comprising using the score of ATTR biomarkers to determine whether the subject from whom the sample was obtained is selected to receive one or more doses of a TTR stabilizer.

[0345] Embodiment 121. The method of embodiment 120, further comprising administering one or more doses of a TTR stabilizer to the subject.

[0346] Embodiment 122. A non-transitory computer-readable medium comprising executable instructions that, when executed, cause a processor to perform operations including the method of any one of embodiments 1 to 121.

[0347] Embodiment 123. (a) one or more ATTR biomarkers, including (i) TnI, (ii) PKM1, (iii) PKM2, (iv) NT-proBNP, (v) RBP4, (vi) TIMP2, (vii) NfL, or (viii) a combination thereof; and (b) one or more anti-ATTR biomarker drugs, including (i) an anti-TnI drug, (ii) an anti-PKM1 drug, (iii) an anti-PKM2 drug, (iv) an anti-NT-proBNP drug, (v) an anti-RBP4 drug, (vi) an anti-TIMP2 drug, (vii) an anti-NfL drug, or (viii) a combination thereof. A composition comprising:

[0348] Embodiment 124. (a) one or more ATTR biomarkers, including TnI, and (b) one or more anti-ATTR biomarker drugs, including anti-TnI drugs A composition comprising:

[0349] Embodiment 125. (a) the one or more ATTR biomarkers include TnI and PKM1; (b) the one or more anti-ATTR biomarker drugs include an anti-TnI drug and an anti-PKM1 drug; The composition of embodiment 124.

[0350] Embodiment 126. (a) the one or more ATTR biomarkers include TnI and PKM2; (b) the one or more anti-ATTR biomarker agents include an anti-TnI agent and an anti-PKM2 agent; The composition of embodiment 124.

[0351] Embodiment 127. (a) the one or more ATTR biomarkers include TnI, PKM1 and PKM2; (b) the one or more anti-ATTR biomarker drugs include an anti-TnI drug, an anti-PKM1 drug, and an anti-PKM2 drug; The composition of embodiment 124.

[0352] Embodiment 128. (a) the one or more ATTR biomarkers further include NT-proBNP, RBP4 or both; (b) the one or more anti-ATTR biomarker drugs further include an anti-NT-proBNP drug, an anti-RBP4 drug, or both; The composition of any one of embodiments 123 to 127.

[0353] Embodiment 129. (a) the one or more ATTR biomarkers further include TIMP2, NfL, or both; (b) the one or more anti-ATTR biomarker drugs further comprise an anti-TIMP2 drug, an anti-NfL drug, or both; The composition of any one of embodiments 123 to 128.

[0354] Embodiment 130. (a) one or more ATTR biomarkers, including NT-proBNP, and (b) one or more anti-ATTR biomarker drugs, including anti-NT-proBNP drugs A composition comprising:

[0355] Embodiment 131. (a) the one or more ATTR biomarkers further comprise: (i) TnI, (ii) PKM1, (iii) PKM2, (iv) RBP4, (v) TIMP2, (vi) NfL, or (vii) a combination thereof; (b) the one or more anti-ATTR biomarker drugs further comprise: (i) an anti-TnI drug, (ii) an anti-PKM1 drug, (iii) an anti-PKM2 drug, (iv) an anti-RBP4 drug, (v) an anti-TIMP2 drug, (vi) an anti-NfL drug, or (vii) a combination thereof; The composition of embodiment 130.

[0356] Embodiment 132. (a) the one or more ATTR biomarkers include NT proBNP, TnI and RBP; (b) the one or more anti-ATTR biomarker drugs include an anti-NT proBNP drug, an anti-TnI drug, and an anti-RBP drug; 132. The composition of embodiment 130 or 131.

[0357] Embodiment 133. The composition of any one of embodiments 123, 124, 126 and 128-132, wherein the one or more ATTR biomarkers do not include PKM1 and the one or more anti-ATTR biomarker agents do not include an anti-PKM1 agent.

[0358] Embodiment 134. The composition of any one of embodiments 123-125 and 128-132, wherein the one or more ATTR biomarkers do not include PKM2 and the one or more anti-ATTR biomarker agents do not include an anti-PKM2 agent.

[0359] Embodiment 135. The composition of any one of embodiments 123, 124, and 128-132, wherein the one or more ATTR biomarkers do not include PKM1 or PKM2, and the one or more anti-ATTR biomarker agents do not include an anti-PKM1 agent or an anti-PKM2 agent.

[0360] Embodiment 136. A kit for detecting TTR-CM, comprising: (a) one or more anti-ATTR biomarker agents, including (i) an anti-TnI agent, (ii) an anti-PKM1 agent, (iii) an anti-PKM2 agent, (iv) an anti-NT-proBNP agent, (v) an anti-RBP4 agent, (vi) an anti-TIMP2 agent, (vii) an anti-NfL agent, or (viii) a combination thereof; and (b) instructions for use. Including the kit.

[0361] Embodiment 137. A kit for detecting TTR-CM, comprising: (a) one or more anti-ATTR biomarker drugs, including an anti-TnI drug; and (b) Instructions for use Kit including:

[0362] Embodiment 138. The kit of embodiment 137, wherein the one or more anti-ATTR biomarker agents comprise an anti-TnI agent and an anti-PKM1 agent.

[0363] Embodiment 139. The kit of embodiment 137, wherein the one or more anti-ATTR biomarker agents comprise an anti-TnI agent and an anti-PKM2 agent.

[0364] Embodiment 140. The kit of embodiment 137, wherein the one or more anti-ATTR biomarker agents comprise an anti-TnI agent, an anti-PKM1 agent, and an anti-PKM2 agent.

[0365] Embodiment 141. The kit of any one of embodiments 137 to 140, wherein the one or more anti-ATTR biomarker drugs further comprise an anti-NT-proBNP drug, an anti-RBP4 drug, or both.

[0366] Embodiment 142. The kit of any one of embodiments 137-141, wherein the one or more anti-ATTR biomarker agents further comprise an anti-TIMP2 agent, an anti-NfL agent, or both.

[0367] Embodiment 143. (a) one or more anti-ATTR biomarker drugs, including an anti-NT-proBNP drug; and (b) Instructions for use Including the kit.

[0368] Embodiment 144. The kit of embodiment 143, wherein the one or more anti-ATTR biomarker drugs further comprise: (i) an anti-TnI drug, (ii) an anti-PKM1 drug, (iii) an anti-PKM2 drug, (iv) an anti-RBP4 drug, (v) an anti-TIMP2 drug, (vi) an anti-NfL drug, or (vii) a combination thereof.

[0369] Embodiment 145. The kit of embodiment 143 or 144, wherein the one or more anti-ATTR biomarker drugs comprise an anti-NT-proBNP drug, an anti-TnI drug, and an anti-RBP drug.

[0370] Embodiment 146. The kit of any one of embodiments 136, 137, 139 and 141-145, wherein the one or more ATTR biomarkers do not include PKM1 and the one or more anti-ATTR biomarker agents do not include an anti-PKM1 agent.

[0371] Embodiment 147. The kit of any one of embodiments 136-138 and 141-145, wherein the one or more ATTR biomarkers do not include PKM2 and the one or more anti-ATTR biomarker agents do not include an anti-PKM2 agent.

[0372] Embodiment 148. The kit of any one of embodiments 136, 137, and 141-145, wherein the one or more ATTR biomarkers do not include PKM1 or PKM2, and the one or more anti-ATTR biomarker agents do not include an anti-PKM1 agent or an anti-PKM2 agent.

[0373] Embodiment 149. The kit of any one of embodiments 136 to 148, further comprising a TTR stabilizer.

[0374] Embodiment 150. The kit of any one of embodiments 136 to 149, wherein the one or more anti-ATTR biomarker drugs comprise one or more antibody drugs.

[0375] Embodiment 151. The kit of any one of embodiments 136 to 150, wherein the one or more anti-ATTR biomarker agents comprise one or more nucleic acid probes.

[0376] Embodiment 152 The kit of embodiment 150 or 151, wherein one or more of the antibody drugs is labeled with a detectable moiety.

[0377] Embodiment 153. The kit of embodiments 150-152, further comprising one or more secondary antibody drugs that specifically bind to the one or more anti-ATTR biomarker antibody drugs.

[0378] Embodiment 154. The kit of embodiment 153, wherein one or more of the one or more anti-ATTR biomarker antibody drugs and / or one or more of the secondary antibody drugs are linked to an enzyme.

[0379] Embodiment 155. The kit of any one of embodiments 136 to 154, further comprising a detection agent.

[0380] Embodiment 156 The kit of embodiment 155, wherein the detection agent is or comprises a substrate for the enzyme.

[0381] Embodiment 157. The kit of embodiment 155, wherein the detection agent is or comprises one or more acridinium ester molecules.

[0382] Embodiment 158. The kit of embodiment 153, wherein one or more of the one or more anti-ATTR biomarker antibody drugs and / or one or more of the secondary antibody drugs are labeled with one or more acridinium ester molecules.

[0383] Embodiment 159. The kit of any one of embodiments 151 to 158, wherein one or more of the anti-ATTR biomarker nucleic acid probes are complementary to one or more nucleotides encoding an ATTR biomarker.

[0384] Embodiment 160. The kit of any one of embodiments 151-159, wherein at least a portion of each anti-ATTR biomarker nucleic acid probe hybridizes to one or more nucleotides encoding an ATTR biomarker.

[0385] Embodiment 161 The kit of any one of embodiments 159 and 160, wherein the nucleotide sequence encoding the one or more ATTR biomarkers is DNA.

[0386] Embodiment 162. The kit of embodiment 161, wherein the DNA is cDNA.

[0387] Embodiment 163 The kit of any one of embodiments 159 and 160, wherein the nucleotide sequence encoding the one or more ATTR biomarkers is RNA.

[0388] Embodiment 164. The kit of any one of embodiments 151-163, wherein one or more of the anti-ATTR biomarker nucleic acid probes are labeled with one or more detection agents.

[0389] Embodiment 165. The kit of embodiment 164, wherein the detection agent indicates the presence of nucleotides encoding one or more ATTR biomarkers.

[0390] Embodiment 166. The kit of any one of embodiments 136 to 165, further comprising one or more control samples.

[0391] Embodiment 167. The kit of embodiment 166, wherein the control sample comprises one or more ATTR biomarker standards.

[0392] Embodiment 168 The kit of embodiment 167, wherein the one or more ATTR biomarker standards comprise recombinant ATTR biomarkers.

[0393] Embodiment 169. The kit of embodiment 167, wherein the one or more ATTR biomarker standards comprise synthetic ATTR biomarker nucleic acids.

[0394] Embodiment 170. Use of a kit according to embodiments 136 to 169 in an in vitro diagnostic assay for diagnosing TTR-CM in a subject. EXAMPLES

[0395] Identifying markers for ATTR amyloidosis This example demonstrates a method for screening human plasma to identify biomarkers indicative of ATTR amyloidosis and TTR-CM.

[0396] Mass spectrometry (MS) was used to identify biomarkers specific for ATTR amyloidosis. Human normal (N=4) and ATTRwt EDTA plasma (N=6) were added (50uL) to prewashed (1x TTBS) goat anti-mouse 96-well ELISA plates and incubated for 1 hour at room temperature. Plates were subsequently washed (3x in 1x TTBS) and residual captured material was extracted and prepared for MS.

[0397] MS results expressed as an Exponentially Modified Protein Abundance Index (emPAI) were generated and data were sorted from highest to lowest emPAI scores. Signal to noise (S / N) ratios were calculated for the mean emPAI scores of ATTRwt / normal scores. The inverse S / N was also calculated. Selected markers were chosen with S / N ratios of 3 or higher (Figure 14). Criteria for marker selection included having multiple consistent score values ​​in pathological material, normal samples or both categories. Missing or null data correspond to values ​​below the detectable limit. For the purpose of generating S / N values, null data were artificially scored with a value of "1". Eight different markers were selected from the list in Table 1 to pursue as possible markers for ATTRwt (Table 6).

[0398] [Table 6] EXAMPLES

[0399] Patient cohorts for biomarker studies This example provides information about the patient cohorts used in the analysis of how the various ATTR biomarkers performed in detecting TTR-CM. As shown in Figure 1, 273 heart failure patients, 46 clinical trial patients, and 20 independent presumed normal donors participated in the study. All individuals in the study were aged 60 years or older and were primarily Caucasian. The number of males and females in each subset differed. The NYHA class of the patients also differed between the subsets. Notably, only a few positive samples with matching negatives were available in the cohort. EXAMPLES

[0400] Summary of test results This example summarizes data obtained from an analysis of how various ATTR biomarkers performed in detecting TTR-CM using the patient cohort described in Example 2. As shown in Figure 2. Only one result was missing across all biomarkers tested. The results for this RBP could not be obtained because there was insufficient sample volume for the necessary retest. Overall, the results of the in vitro diagnostic assays were based on one test per sample; the average of two test replicates was utilized to account for the higher variability of studies using only the assay. EXAMPLES

[0401] Classification performance of single biomarkers This example illustrates the evaluation of exemplary ATTR biomarkers described herein for their ability to be used as single biomarkers for the detection of TTR-CM. The results show that certain ATTR biomarkers described herein can work at sufficient sensitivity and / or specificity levels to allow them to be used alone. However, this example also shows that not all ATTR biomarkers met the criteria for both sensitivity and specificity when used as single biomarkers to evaluate TTR-CM. As shown in Figures 3-6, TnI met both the sensitivity and specificity criteria, suggesting that TnI may be useful as a single biomarker for the detection of TTR-CM. The remaining ATTR biomarkers evaluated did not meet both the sensitivity and specificity criteria set for use as single biomarkers. These biomarkers were then evaluated in combination to determine whether combinations of ATTR biomarkers could achieve improved sensitivity and specificity when detecting TTR-CM. EXAMPLES

[0402] Single biomarker NYHA class response screening This example shows the evaluation of the effect of biomarkers when considering each NYHA class.As shown in Figure 7, the effect of all biomarkers on NYHA class was modeled and p-values ​​for significance test were reported.The data showed that the effect of biomarkers was statistically significant for PKM, TnI, RBP4, RBP and NT-proBNP.This shows that these ATTR biomarkers can be useful for detecting TTR-CM, despite the fact that none of these biomarkers met the criteria of sensitivity and specificity as described above in Example 4. EXAMPLES

[0403] Performance Results This example illustrates screening of generalized regression fits to process data obtained from a combination of biomarkers. Main effects and interactions were screened, and the best fit with up to four biomarkers was selected for further evaluation.

[0404] An adaptive double lasso generalized regression method with leave-one-out validation using a complete response surface as input was expected to select a near-optimal model using a minimal number of effects in a small dataset containing several correlated biomarkers. As shown in Figure 8, depending on the method used, different subsets of biomarkers were found to be informative. However, a limit of four biomarkers was determined based on practical considerations. Several subsets of biomarkers and predictive models were generated. These biomarkers were further evaluated using additional machine learning including PKM, TnI, DCN, TIMP2 and NT-proBNP.

[0405] Further model screening was performed using a combination of PKM, TnI, DCN, TIMP2 and NT-proBNP. As shown in Figure 9, the bootstrap forest model was found to provide the best results compared to other machine learning methods. The neural network model also provided good results, but with more false negatives.

[0406] These results indicate that a triple or quadruple biomarker combination, including TnI and PKM, especially with NT-proBNP and / or RBP4, can detect TTR-CM with high sensitivity and specificity. EXAMPLES

[0407] Identification of PKM and TIMP2 as elevated biomarkers in patients with ATTR amyloidosis This example shows that two biomarkers (PKM and TIMP2) were identified that individually and in combination showed elevated plasma levels in ATTRwt patients compared to normal human control samples.

[0408] Selected biomarkers in Table 2 were tested by ELISA for immunoreactivity in normal human plasma and ATTR amyloidosis plasma (Table 7).

[0409] [Table 7]

[0410] [Table 8]

[0411] Signal-to-noise (S / N) ratios (calculated for each biomarker as the raw absorbance value of cardiomyopathic (CM) or normal (N) samples divided by the mean of normal samples) were determined for each biomarker assay. Cutoff values ​​were set as the highest S / N value of normal plasma samples for each biomarker assay, except for assays for ILK and PKM, which were excluded because their respective highest normal samples were greater than the mean + (4 × standard deviation).

[0412] Using the S / N values ​​and comparing them to the cutoff values ​​for each biomarker, each sample (i.e., CM or N) was scored as positive (>cutoff) or negative (<cutoff) with a score of 1 or 0, respectively (Table 8). The performance ratio of each biomarker, including sensitivity, specificity, and accuracy, was also determined by the following calculations: % sensitivity = number of CM samples scored positive / total CM samples; % specificity = (1-number of normal samples scored positive) x 100; % accuracy = (number of reported positive CM samples + number of reported negative normal samples) / total samples tested (CM and N) x 100.

[0413] [Table 9-1] [Table 9-2]

[0414] The performance scores of the various biomarkers tested showed high sensitivity, specificity and accuracy for two individual markers, TIMP2 (sensitivity 72%, specificity 100%, accuracy 87%) and PKM (sensitivity 83%, specificity 97%, accuracy 90%). When the two markers, TIMP2 and PKM, were combined across the same set of samples, the sensitivity, specificity and accuracy were significantly improved (sensitivity 93%, specificity 97%, accuracy 95%), suggesting that the combined TIMP2 and PKM can act as a sensitive and specific marker for identification of ATTRwt plasma. EXAMPLES

[0415] Biomarker threshold calculation This example demonstrates the identification of thresholds for biomarkers of ATTR amyloidosis.

[0416] A total of N=30 diagnosed TTR-CM patient plasma samples and N=30 normal donor samples were evaluated for expression of PKM, TIMP2, LIMS1, C3, and A11. Sensitivity, specificity, and accuracy were calculated as described above. Sensitivity and specificity calculations assumed no undiagnosed TTR-CM donors. Optimized detection cutoffs for each assay were determined by maximizing sensitivity while retaining approximately 100% specificity (Table 9 and Figure 10).

[0417] The disclosed methods can be performed manually or can be automated. For example, the disclosed methods can use the ADVIA CENTAUR® Immunoassay System or the ATELLICA@™. For example, the system can be automated to perform the following operations: 1. Dispense the sample into a cuvette. 2. A buffer solution containing a solid support to which an anti-human IgM antibody is bound is dispensed and incubated, for example, at 37° C. for 18.25 minutes. 3. Detach / separate the solid support and aspirate the cuvette and wash with washing reagent. 4. Dispense buffer containing chemiluminescently tagged NS1 antigen and incubate, for example, at 37° C. for 18 minutes. 5. Detach / separate the solid support and aspirate the cuvette and wash with washing reagent. 6. Dispense the chemiluminescent reagent to initiate the chemiluminescent reaction. 7. Report the results according to the options selected by the user.

[0418] In certain embodiments, the disclosed immunoassays may be suitable for use in an ADVIA CENTAUR immunoassay system (Siemens Healthcare, AG) and / or the ADVIA CENTAUR immunoassay system may be automated to perform the operations described above.

[0419] [Table 10] EXAMPLES

[0420] Patient evaluation A patient at risk for TTR-CM is tested for the level of each ATTR biomarker in an ATTR biomarker combination, for example as disclosed herein in Tables 2-5. A blood sample is drawn from the patient and the levels are measured using a Siemens Atellica® system or a Siemens Advia Centaur® system to obtain an ATTR biomarker profile for the patient. Patient demographic factors and image-based biomarkers are taken into account, if necessary. The patient's ATTR biomarker profile is used to calculate an ATTR biomarker score. The patient is categorized as having TTR-CM based on the ATTR biomarker score. Administration of a TTR stabilizer or other cardiac medication is recommended. EXAMPLES

[0421] Patient evaluation A patient at risk for TTR-CM is tested for the level of each ATTR biomarker in an ATTR biomarker combination, e.g., as disclosed herein in Tables 2-5. A blood sample is drawn from the patient and the levels are measured using a Siemens Atellica® system or a Siemens Advia Centaur® system to obtain an ATTR biomarker profile for the patient. Patient demographic factors and image-based biomarkers are taken into account, if necessary. The patient's ATTR biomarker profile is compared to a reference ATTR biomarker profile. Based on the comparison, the patient is categorized as having TTR-CM. Administration of a TTR stabilizer or other cardiac medication is recommended. EXAMPLES

[0422] Patient evaluation A patient at risk for TTR-CM is tested for the level of each ATTR biomarker in the ATTR biomarker combination, e.g., as disclosed herein in Tables 2-5. A blood sample is drawn from the patient and the levels are measured using a Siemens Atellica® system or a Siemens Advia Centaur® system to obtain an ATTR biomarker profile for the patient. Patient demographic factors and image-based biomarkers are taken into account, if necessary. The patient's ATTR biomarker profile is used to calculate an ATTR biomarker score. The patient is categorized as at risk for having TTR-CM based on the ATTR biomarker score. Further cardiomyopathy testing is recommended. EXAMPLES

[0423] Patient evaluation A patient at risk for TTR-CM is tested for the level of each ATTR biomarker in the ATTR biomarker combination, e.g., as disclosed herein in Tables 2-5. A blood sample is drawn from the patient and the levels are measured using a Siemens Atellica® system or a Siemens Advia Centaur® system to obtain an ATTR biomarker profile for the patient. Patient demographic factors and image-based biomarkers are taken into account, if necessary. The patient's ATTR biomarker profile is compared to a reference ATTR biomarker profile. Based on the comparison, the patient is categorized as at risk for having TTR-CM. Further cardiomyopathy testing is recommended. EXAMPLES

[0424] Group evaluation 1000 patients at risk for TTR-CM are tested for the levels of each ATTR biomarker in the ATTR biomarker combination, e.g., as disclosed herein in Tables 2-5. A blood sample is drawn from the patient and the levels are measured using a Siemens Atellica® system or a Siemens Advia Centaur® system to obtain an ATTR biomarker profile for the patient. Patient demographic factors and image-based biomarkers are taken into account, if necessary. The patient's ATTR biomarker profile is used to calculate a score for each ATTR biomarker. A subset of patients is categorized as at risk for having TTR-CM. Further cardiomyopathy testing is recommended. EXAMPLES

[0425] Clinical utility of non-invasive and accurate TTR-CM This example demonstrates the benefits of providing the public with a non-invasive TTR-CM assay that is both sensitive and specific and easy to comply with. This example shows that reluctance to undergo invasive cardiomyopathy testing is common and that this can have serious health consequences if TTR-CM is not detected early.

[0426] The patient exhibited symptoms of TTR-CM but refused a cardiac biopsy. The patient's primary care physician requested an evaluation of the patient's levels of each ATTR biomarker in a combination of ATTR biomarkers, such as those disclosed herein in Tables 2-5. The results indicated that the patient was at high risk for TTR-CM. The patient consulted with his physician and was persuaded to schedule further cardiomyopathy testing to clarify the basis of TTR-CM.

[0427] References 1. Gertz, M.A., Benson, M.D., Dyck, P.J., Grogan, M. Coelho, T., Crus, M., Berk, J.L., Plante-Bordeneuve, V., Schmidt, H.H., Merlini, G. Diagnosis, Prognosis, and Therapy of Transthyretin Amyloidosis. JACC 66:2452-2466 (2015). 2. Ashley 2004 3. Krishnamurthy, R., Cheong, B., Muthypillai, R. Tools for cardiovascular magnetic resonance imaging. Current Cardiology Reviews, 9:185-190, 2013. 4. Doltra, A., Amundsen, B.H., Gebker, R., Fleck, E., Kelle, S. Emerging concepts for myocardial late gadolinium enhancement MRI. Curr Cardiol Rev. 9(3):185-90, 2013. 5. Bokhari, S., Castano, A., Pozniakoff, T., Deslisle, S., Latif, F., Maurer, M.S., (99m)Tc-pyrophosphate scintigraphy for differentiating light-chain cardiac amyloidosis from the transthyretin-related familial and senile cardiac amyloidoses. Circ Cardiovasc Imaging. 6(2):195-201, 2013. 6. Crotty, TB Li, CY, Edwards, WD, Suman, VJ Amyloidosis and endomyocardial biopsy: Correlation of extent and pattern of deposition with amyloid immunophenotype in 100 cases. Cardiovascular Pathology 4:39-42, 1995. 7. Arvanitis, M. et al. Identification of Transthyretin Cardiac Amyloidosis Using Serum Retinol-Binding Protein 4 and a Clinical Prediction Model. JAMA Cardiol., 2017.

[0428] All publications and patents cited in this disclosure, including those listed above, are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0429] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. The scope of the disclosure is not intended to be limited to the above description, but rather is as set forth in the following claims.

Claims

1. 1. A method, comprising detecting a level of each of two or more transthyretin amyloidosis (ATTR) biomarkers in a sample, the two or more ATTR biomarkers comprising: (i) Troponin I (TnI); (ii) pyruvate kinase muscle isoform 1 (PKM1); (iii) pyruvate kinase muscle isoform 2 (PKM2); (iv) N-terminal-prohormone B-type natriuretic peptide (NT-proBNP); (v) retinol binding protein 4 (RBP4); (vi) tissue inhibitor of metalloproteinase 2 (TIMP2); (vii) neurofilament light chain (NfL), or (viii) combinations thereof A method comprising:

2. A method, the method comprising detecting respective levels of two or more ATTR biomarkers in a sample, the two or more ATTR biomarkers comprising (i) TnI and PKM1, (ii) TnI and PKM2, or (iii) TnI, PKM1 and PKM2.

3. 1. A method, comprising: (a) detecting the respective levels of two or more ATTR biomarkers in a sample obtained from a subject to obtain an ATTR biomarker profile; and (b) computing an ATTR biomarker score using the ATTR biomarker profile. and the two or more ATTR biomarkers include (i) Troponin I (TnI); (ii) pyruvate kinase muscle isoform 1 (PKM1); (iii) pyruvate kinase muscle isoform 2 (PKM2); (iv) N-terminal-prohormone B-type natriuretic peptide (NT-proBNP); (v) retinol binding protein 4 (RBP4); (vi) tissue inhibitor of metalloproteinase 2 (TIMP2); (vii) neurofilament light chain (NfL), or (viii) combinations thereof Including, Using the ATTR biomarker profile to compute an ATTR biomarker score comprises applying an algorithm to the ATTR biomarker profile to compute an ATTR biomarker score, the algorithm being derived from a decision tree method, a neural boosting method, a bootstrap forest method, a boosted tree method, or a support vector machine method. method.

4. The method of claim 3 , wherein the subject is a human subject.

5. The method of any one of claims 1 to 4, wherein the sample comprises blood, serum, plasma or cardiac tissue.

6. 4. The method of claim 3, wherein the score of the ATTR biomarker is used to determine whether the subject from which the sample was obtained is at risk for or suffers from transthyretin amyloid cardiomyopathy (TTR-CM).

7. The method of claim 3, wherein the score of the ATTR biomarker is used to diagnose the subject with TTR-CM.

8. 4. The method of claim 3, further comprising using the ATTR biomarker score to determine whether the subject from whom the sample was obtained is selected for one or more cardiomyopathy tests.

9. 4. The method of claim 3, further comprising using the score of the TTR biomarkers to determine whether the subject from whom the sample was obtained is selected to receive one or more doses of an TTR stabilizing agent.

10. The method of any one of claims 1 to 4 and 6 to 9, wherein the two or more ATTR biomarkers do not include PKM1.

11. The method of any one of claims 1 to 4 and 6 to 9, wherein the two or more ATTR biomarkers do not include PKM2.

12. The method of any one of claims 1 to 4 and 6 to 9, wherein the two or more ATTR biomarkers do not include PKM1 or PKM2.

13. The method of any one of claims 1 to 4 and 6 to 9, wherein the two or more ATTR biomarkers do not include SMOC-2.

14. The method of any one of claims 1 to 4 and 6 to 9, wherein the two or more ATTR biomarkers do not include DCN.

15. Claims 1 to 4, wherein the two or more ATTR biomarkers do not include SMOC-2 or DCN. and the method according to any one of claims 6 to 9.

16. A non-transitory computer readable medium comprising executable instructions that, when executed, cause a processor to perform operations including the method of any one of claims 1-4 and 6-9.

17. 1. A composition comprising: (a) (i) TnI, (ii) PKM1, (iii) PKM2, (iv) NT-proBNP, (v) RBP4, (vi) TIMP2, (vii) NfL, or (viii) combinations thereof and one or more ATTR biomarkers, (b)(i) an anti-TnI drug; (ii) an anti-PKM1 drug, (iii) anti-PKM2 drugs, (iv) anti-NT-proBNP drugs, (v) anti-RBP4 drugs, (vi) anti-TIMP2 drugs, (vii) an anti-NfL drug, or (viii) combinations thereof One or more anti-ATTR biomarker drugs comprising A composition comprising:

18. A kit for detecting TTR-CM, comprising: (a)(i) an anti-TnI drug; (ii) an anti-PKM1 drug, (iii) anti-PKM2 drugs, (iv) anti-NT-proBNP drugs, (v) anti-RBP4 drugs, (vi) anti-TIMP2 drugs, (vii) an anti-NfL drug, or (viii) combinations thereof and one or more anti-ATTR biomarker drugs comprising (b) Instructions for use. Including the kit.

19. A kit for detecting TTR-CM, comprising: (a) one or more anti-ATTR biomarker drugs, including (i) an anti-TnI drug and an anti-PKM1 drug, (ii) an anti-TnI drug and an anti-PKM2 drug, or (iii) an anti-TnI drug, an anti-PKM1 drug, and an anti-PKM2 drug; and (b) Instructions for use. Including the kit.

20. A kit comprising: (a) one or more anti-ATTR biomarker drugs, including anti-NT-proBNP drugs, anti-TnI drugs, and anti-RBP4 drugs; and (b) Instructions for use. Including the kit.

21. The kit of any one of claims 18 to 20, wherein the one or more anti-ATTR biomarker drugs comprise one or more antibody drugs.

22. 22. The kit of claim 21, wherein one or more of the antibody drugs is labeled with a detectable moiety.

23. The kit of any one of claims 18 to 20, further comprising one or more control samples.

24. 24. The kit of claim 23, wherein the control sample comprises one or more ATTR biomarker standards.

25. Use of the kit according to any one of claims 18 to 20 in an in vitro diagnostic assay for diagnosing TTR-CM in a subject.

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