Structure-based probes for the detection of transthyretin amyloid fibrils and aggregates

Abstract

The disclosure of this specification relates to a polypeptide probe that can be used to detect transthyretin (TTR) oligomers or fibrils in patient samples obtained from subjects having wild-type and mutant TTR alleles. Also provided are methods of using the provided probe to diagnose a subject with a TTR-related disease or condition, or to monitor the efficacy of a therapeutic agent administered to treat a TTR-related disease or condition.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 352,521, filed Jun. 15, 2022, and U.S. Provisional Application No. 63 / 382,122, filed Nov. 3, 2022, the disclosures of each of which are hereby incorporated by reference in their entireties.

[0002] Approval of Government Support This invention was made with government support under Grant No. HL163810 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing This application is being electronically submitted in XML format and includes a sequence listing that is hereby incorporated by reference in its entirety for all purposes. This XML copy, created on Jun. 14, 2023, is named 106546 - 761784(UTSD - 4057 - WO).xml and is 41 kilobytes in size.

Background Art

[0004] Background 1. Field

[0005] The concept of the present invention relates to compositions containing peptide probes for the detection and quantification of transthyretin amyloid fibrils and aggregates.

[0006] 2. Consideration of Related Fields

[0007] Amyloid diseases are characterized by the aggregation of certain proteins into amyloid fibrils that can then function as seeds to induce further aggregation of the parent protein. One major protein that can tend to aggregate is transthyretin (TTR). Transthyretin (TTR) is a 55 kDa tetrameric protein that transports retinol-binding protein (RBP) and thyroxine (T4) in the blood and cerebrospinal fluid. Amyloid aggregation of TTR occurs by dissociation of the tetrameric TTR into monomers; these partially unfold into amyloidogenic intermediates and self-associate into soluble oligomers and amyloid aggregates. Familial point mutations are known to destabilize the tetramer, leading to more rapid dissociation and resulting amyloid aggregation.

[0008] Two diseases are associated with transthyretin amyloidosis (ATTR). Wild-type amyloidosis is a late-onset disease in which fibrils composed of wild-type (WT) TTR weaken the myocardium. Postmortem studies have shown that 25% of individuals over 80 years old have WT ATTR in the heart. Furthermore, variant ATTR amyloidosis is a genetic condition with a variable clinical picture. Both amyloidoses are lethal disorders characterized by extracellular deposition of TTR amyloid fibrils in various tissues, such as the kidney, eye, gastrointestinal tract, and skin. Some of the most harmful depositions are in the heart and peripheral nerves, leading to cardiomyopathy and polyneuropathy. Hereditary amyloidotic polyneuropathy includes a set of mutations, such as L55P and V30M, and results in progressive sensorimotor and autonomic neuropathy. Hereditary amyloidotic cardiomyopathy includes the mutation V122I and causes protein deposition in heart tissue.

[0009] Early detection of amyloid fibrils or transthyretin aggregates in patients is important for the patient's outcome, especially in the case of sporadic amyloidosis where the patient has no known mutations associated with TTR aggregation. Therefore, there is a great need for suitable detection factors or probes for identifying and / or quantifying TTR aggregation in larger patient populations. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0010] ABSTRACT The present disclosure is at least partly based on the discovery of peptide probes that bind tightly to transthyretin aggregates or oligomers. These can be used for the labeling and detection of transthyretin.

[0011] Aspects of the present disclosure provide a polypeptide probe comprising a first peptide comprising the sequence HVAHPFVEFTE (SEQ ID NO: 1) and a second peptide comprising the sequence SYVTNPTSYAVT (SEQ ID NO: 2), wherein the first and second peptides are covalently linked via a linker peptide and the polypeptide probe further comprises a detectable label.

[0012] In various aspects, the first and second peptides of the polypeptide probe bind simultaneously to two different strands of a transthyretin fiber or aggregate. In some aspects, the first peptide binds to a first strand of a transthyretin fiber or aggregate and the second peptide binds to a second strand of the transthyretin fiber or aggregate, and the first strand and the second strand are different. For example, the two strands bound by the first and / or second peptides can be the "F" strand or the "H" strand.

[0013] In any of the aspects of the present disclosure, the linker peptide may include the sequences GGGSTE (SEQ ID NO: 3), EAAAK (SEQ ID NO: 4), PAPAP (SEQ ID NO: 5), or GGGGGG (SEQ ID NO: 6). In various aspects, the linker peptide includes GGGSTE (SEQ ID NO: 3).

[0014] In any of the aspects of the present disclosure, the polypeptide probe may further include an epitope tag that facilitates the lysis, manipulation, and / or purification of the polypeptide. In some aspects, the epitope tag includes a peptide that increases the affinity of the polypeptide for an affinity column. For example, the epitope tag may include multiple histidine or lysine residues. In other aspects, the epitope tag includes a peptide consisting of the sequence DYKDDDDK (SEQ ID NO: 7) or YPYDVPDYA (SEQ ID NO: 8). In some aspects, the epitope tag increases the solubility of the polypeptide. For example, in some aspects, the epitope tag may include multiple arginine residues.

[0015] In any of the aspects of the present disclosure, the detectable label is covalently linked to the N-terminus of the first peptide or the C-terminus of the second peptide. In some aspects, the detectable label is covalently linked to the polypeptide probe via a linker (e.g., aminohexanoic acid (Ahx)). In various aspects, the detectable label includes tetramethylrhodamine (TAMRA) or fluorescein isothiocyanate (FITC).

[0016] In any of the aspects of the present disclosure, the polypeptide probe may comprise the amino acid sequence RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 9). In other aspects, the polypeptide probe may comprise an amino acid sequence selected from YPYDVPDYARRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 10), DYKDDDDKRRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 11), or HHHHHHRRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 12).

[0017] In various embodiments, the polypeptide probe can be selected from TAMRA-YPYDVPDYA-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 14), TAMRA-DYKDDDDK-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 15), TAMRA-HHHHHH-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 16), FITC-Ahx-YPYDVPDYA-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 17), FITC-Ahx-DYKDDDDK-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 18), FITC-Ahx-HHHHHH-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 19), and FITC-Ahx-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 20), where TAMRA is tetramethylrhodamine, FITC is fluorescein isothiocyanate (FITC), and Ahx is an aminohexanoic acid linker. In some embodiments, the polypeptide probe can be selected from FITC-Ahx-HHHHHH-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 19), FITC-Ahx-YPYDVPDYA-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 17), or FITC-Ahx-DYKDDDDK-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 18), where FITC is fluorescein isothiocyanate (FITC) and Ahx is an aminohexanoic acid linker. In some embodiments, the polypeptide probe is FITC-Ahx-HHHHHH-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 19), where FITC is fluorescein isothiocyanate (FITC) and Ahx is an aminohexanoic acid linker.

[0018] Also provided herein are pharmaceutical compositions comprising the polypeptide probes provided herein and a pharmaceutically suitable carrier or excipient.

[0019] A method of detecting an oligomer, aggregate or fiber of transthyretin in a sample, comprising: (a) contacting the sample with a polypeptide probe provided herein; (b) binding the polypeptide probe to any oligomer, aggregate or fiber of transthyretin in the sample; and (c) detecting a complex comprising the polypeptide and the oligomer, aggregate or fiber of transthyretin, wherein the presence of the complex correlates with the presence of the oligomer, aggregate or fiber of transthyretin in the sample. Also provided herein. In some embodiments, the sample is obtained from a subject having or suspected of having transthyretin amyloidosis. In further embodiments, the sample can include a blood sample (e.g., a plasma or serum sample), a tissue sample or a cerebrospinal fluid sample. In various embodiments, the tissue sample can include a transthyretin-containing tissue (e.g., tissue obtained from a heart biopsy, a fat biopsy, a nerve biopsy, a gastrointestinal biopsy and / or a salivary gland biopsy). In still other embodiments, the sample is obtained from a subject having a wild-type allele of the gene encoding transthyretin. In other embodiments, the sample is obtained from a subject having a variant allele of the gene encoding transthyretin.

[0020] A method of diagnosing a subject with a TTR-related disorder or disease, comprising: (a) detecting an oligomer, fiber or molecule of transthyretin in a sample obtained from the subject according to the method provided herein; and (b) diagnosing the subject with a TTR-related disorder or disease when an oligomer, fiber or molecule of transthyretin is detected in the sample. Also provided herein. In various embodiments, the TTR-related disorder or disease can include ATTR amyloidosis. In some embodiments, the subject is diagnosed with a TTR-related disorder or disease when the level of the oligomer, fiber or molecule of transthyretin detected in the sample exceeds a threshold.

[0021] A further aspect of the present disclosure relates to a method for determining whether a subject is at risk of TTR aggregation. In various aspects, these methods may include: (a) detecting a transthyretin oligomer, fiber or molecule in a sample obtained from a subject according to the methods provided herein; and (b) identifying the subject as being at risk of TTR aggregation if a transthyretin oligomer, fiber or molecule is detected in the sample. In some aspects, the subject is determined to be at risk of TTR aggregation if the level of transthyretin oligomer, fiber or molecule detected in the sample exceeds a threshold.

[0022] In other aspects, a method for monitoring the effectiveness of a therapeutic agent administered to a subject for treating a TTR-related disorder or disease is provided, the method including: (a) detecting an oligomer, aggregate or fiber of transthyretin in a first sample obtained from the subject according to the methods provided herein; (b) administering the therapeutic agent to the subject; and (c) detecting an oligomer, aggregate or fiber of transthyretin in a second sample obtained from the sample after the therapeutic agent has been administered according to any of the methods provided herein, wherein the therapeutic agent is determined to be effective if fewer oligomers, aggregates and / or fibers of transthyretin are detected in the second sample compared to the first sample. In various aspects, these methods may further include monitoring more than one dose of the therapeutic agent to identify an effective amount of the therapeutic agent, the effective amount of the therapeutic agent resulting in the greatest reduction in the detection of oligomers, aggregates and / or fibers of transthyretin in the second sample compared to the first sample. In various aspects, the subject has been diagnosed with a TTR-related disorder or disease according to the methods provided herein.

[0023] In other aspects, provided is a method of treating a subject in need thereof for a TTR-related disorder or disease. In various aspects, the method comprises diagnosing the subject with a TTR-related disorder or disease by detecting TTR oligomers, aggregates and / or fibrils using the polypeptide probes disclosed herein, and then administering to the subject an effective amount of a therapeutic agent. In some aspects, the method may further comprise determining an effective amount of the therapeutic agent according to the methods herein.

[0024] In any of the foregoing or related aspects, the TTR-related disorder or disease includes ATTR amyloidosis.

[0025] In any of the foregoing or related methods, the therapeutic agent includes an inhibitor of transthyretin expression and / or aggregation. For example, in some aspects, the therapeutic agent may include a small molecule, a gene silencer or an antibody. In some aspects, the therapeutic agent includes tafamidis.

[0026] In any of the foregoing or related methods, the subject may have or be suspected of having a condition or characteristic that predisposes the subject to TTR aggregation. For example, the subject may have carpal tunnel, be elderly, be an athlete, have heart failure with preserved ejection fraction (HFpEF), carry a mutation in the TTR gene, or any combination thereof. In some aspects, the subject has or is suspected of having transthyretin amyloidosis. In still further aspects, the subject may have a wild-type allele of the gene encoding transthyretin. In other aspects, the subject may have a variant allele of the gene encoding transthyretin.

[0027] The following drawings form a part of this specification and are included to further demonstrate certain aspects of the present disclosure that can be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein. Embodiments of the present disclosure are shown by way of example, where like reference numerals indicate similar elements.

Brief Description of the Drawings

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[0054] Detailed Description The following detailed description refers to the accompanying drawings that illustrate various embodiments of the present disclosure. The drawings and description are intended to describe the aspects and embodiments of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components may be utilized and changes may be made without departing from the scope of the present disclosure. Accordingly, the following description should not be construed in a limiting sense. The scope of the present disclosure is defined only by the appended claims together with the full scope of equivalents to which such claims are entitled.

[0055] The present disclosure is at least partly based on the discovery of a structure-based peptide detection probe that can selectively detect transthyretin fibers formed from wild-type or mutant transthyretin. I. Technical Terms

[0056] The terms and technical terms used in this specification are for the purpose of explanation and should not be regarded as limiting. For example, the use of singular terms, such as "a", is not intended to limit the number of items. Also, the use of relative terms such as, but not limited to, "top", "bottom", "left", "right", "upper", "lower", "down", "up", and "side" is used in the description for clarity by specifically referring to numbers and is not intended to limit the present disclosure or the appended claims.

[0057] Furthermore, since the present disclosure is susceptible to many different forms of embodiments, the present disclosure is intended to be regarded as an example of the principles of the present disclosure and is not intended to limit the present disclosure to the specific embodiments shown and described. Any one of the features of the present disclosure can be used separately or in combination with any other feature. References to terms such as "embodiment (singular)", "embodiments (plural)", etc. in the description mean that the feature(s) and / or feature(s) being referred to are included in at least one aspect of the description. Separate references to terms such as "embodiment (singular)", "embodiments (plural)", etc. in the description do not necessarily refer to the same embodiment and are not mutually exclusive unless stated so and / or unless it is readily apparent to those skilled in the art from the description. For example, features, structures, processes, steps, actions, etc. described in one embodiment may or may not be included in other embodiments. Accordingly, the present disclosure may also include various combinations and / or integrations of the embodiments described herein. Furthermore, not all aspects of the present disclosure described herein are essential for its implementation. Similarly, other systems, methods, features, and advantages of the present disclosure will be apparent or will become apparent to those skilled in the art upon examination of the drawings and description. All such additional systems, methods, features, and advantages are included within this description, are within the scope of the present disclosure, and are intended to be encompassed by the claims.

[0058] As used herein, the term "about" can mean a relative value with respect to the recited value, e.g., for amounts, dosages, temperatures, times, percentages, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1%.

[0059] The terms "comprising", "including", "encompassing" and "having" are used interchangeably in this disclosure. The terms "comprising", "including", "encompassing" and "having" mean including the things so described but not necessarily limited to them.

[0060] As used herein, the terms "or" and "and / or" should be construed to include either one, or any combination, including all. Thus, "A, B or C" or "A, B and / or C" means any of the following: "A", "B" or "C"; "A and B"; "A and C"; "B and C"; "A, B and C". An exception to this definition occurs only when the combination of elements, functions, steps or acts is mutually exclusive in some way.

[0061] As used herein, the terms "treat", "treating", "treatment", etc., unless otherwise indicated, can refer to reversing, reducing, inhibiting the process of, or preventing a disease, disorder or condition to which such terms are applicable, or one or more symptoms of such disease, disorder or condition, and can include administration of any of the compositions, pharmaceutical compositions or dosage forms described herein for preventing the onset of symptoms or complications, or for reducing symptoms or complications, or for eliminating a condition or disorder.

[0062] The terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers, either in single-stranded or double-stranded form. Unless specifically limited, this term includes nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0063] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute the sequence of a protein or peptide. Polypeptides include any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, this term refers to both short chains, which are commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.

[0064] It should also be understood that, unless the contrary is clearly indicated, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. II. Compositions

[0065] The present disclosure provides compositions for detecting TTR oligomers or aggregates. In various embodiments, these compositions include at least one polypeptide probe comprising a first peptide and a second peptide, the first and second peptides having an affinity for TTR oligomers or aggregates, covalently linked via a linker peptide, and the polypeptide being covalently linked at its N-terminus or C-terminus to a detectable label. 1. Components of the Polypeptide Probe a) Transthyretin-Binding Peptide

[0066] Transthyretin fibers are known to have multiple structural domains identified in the art as A - G chains. In various embodiments, the first and / or second peptide of the polypeptide probe can bind to any of these chains. In various embodiments, the first and second peptides of the polypeptide probe can bind to different chains (e.g., the "F" chain or the "H" chain). In various embodiments, the first and second peptides of the polypeptide probe can simultaneously bind to different (separate) chains of the TTR oligomer / aggregate. In some embodiments, the first peptide of the polypeptide probe can bind to a region (e.g., a chain or loop) of the TTR fiber comprising a peptide selected from VAVHVF (SEQ ID NO: 22, B chain), IYKVEI (SEQ ID NO: 23, E chain), KALGIS (SEQ ID NO: 24, loop connecting E and F chains), AEVVFT (SEQ ID NO: 25; F chain), YTIAAL (SEQ ID NO: 26, G chain), TIALLS (SEQ ID NO: 27, G chain) or TAVVTN (SEQ ID NO: 28, H chain). In some embodiments, the first peptide of the polypeptide probe can bind to the F chain or the H chain (e.g., the chain comprising the peptide AEVVFT (SEQ ID NO: 25) or TAVVTN (SEQ ID NO: 28)). In some embodiments, the first peptide of the polypeptide probe can bind to the F chain. In some embodiments, the first peptide of the polypeptide probe can bind to the H chain. In some embodiments, the second peptide of the polypeptide probe can bind to a region (e.g., a chain or loop) of the TTR fiber comprising a peptide selected from VAVHVF (SEQ ID NO: 22, B chain), IYKVEI (SEQ ID NO: 23, E chain), KALGIS (SEQ ID NO: 24, loop connecting E and F chains), AEVVFT (SEQ ID NO: 25; F chain), YTIAAL (SEQ ID NO: 26, G chain), TIALLS (SEQ ID NO: 27, G chain) or TAVVTN (SEQ ID NO: 28, H chain). In some embodiments, the second peptide of the polypeptide probe can bind to the F chain or the H chain (e.g., the chain comprising the peptide AEVVFT (SEQ ID NO: 25) or TAVVTN (SEQ ID NO: 28)). In some embodiments, the second peptide of the polypeptide probe can bind to the F chain. In some embodiments, the second peptide of the polypeptide probe can bind to the H chain.In some embodiments, the first peptide of the polypeptide probe binds to the F chain, and the second peptide of the polypeptide probe binds to the H chain. In some embodiments, the first peptide of the polypeptide probe binds to the H chain, and the second peptide of the polypeptide probe binds to the F chain.

[0067] In any of the polypeptide probes described herein, the first peptide and / or the second peptide may comprise HVAHPFVEFTE (SEQ ID NO: 1) and / or SYVTNPTSYAVT (SEQ ID NO: 2). For example, in some embodiments, the first peptide may comprise or consist of HVAHPFVEFTE (SEQ ID NO: 1). In some embodiments, the first peptide may comprise or consist of SYVTNPTSYAVT (SEQ ID NO: 2). In other embodiments, the second peptide may comprise or consist of HVAHPFVEFTE (SEQ ID NO: 1). In other embodiments, the second peptide may comprise or consist of SYVTNPTSYAVT (SEQ ID NO: 2).

[0068] In some embodiments, the first peptide comprises or consists of HVAHPFVEFTE (SEQ ID NO: 1), and the second peptide comprises or consists of SYVTNPTSYAVT (SEQ ID NO: 2). In other embodiments, the first peptide comprises or consists of SYVTNPTSYAVT (SEQ ID NO: 2), and the second peptide comprises or consists of HVAHPFVEFTE (SEQ ID NO: 1). b) Linker

[0069] The first and second peptides of the polypeptide probe may be covalently linked via a flexible peptide linker. Suitable linkers may include glycine-rich linkers (e.g., GGGSTE, SEQ ID NO: 3). Other suitable linkers include EAAAK (SEQ ID NO: 4), PAPAP (SEQ ID NO: 4) or GGGGGG (SEQ ID NO: 6). Other suitable flexible linkers are known in the art. c) Epitope tag

[0070] In some embodiments, the polypeptide probe further comprises one or more epitope tags. The epitope tag can, in some embodiments, be used to facilitate purification and concentration of the polypeptide probe (e.g., from an affinity column). In other embodiments, the epitope tag can increase the solubility of the polypeptide probe. In some embodiments, the epitope tag can include an amino acid sequence that increases peptide solubility (e.g., multiple arginine residues); and / or an amino acid sequence that facilitates monitoring or manipulation of the peptide (e.g., multiple lysine and / or histidine residues). In some embodiments, the multiple arginine, lysine, and / or histidine residues include 3 to 12 arginine, lysine, and / or histidine residues (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 arginine, lysine, and / or histidine residues). In some embodiments, the epitope tag includes DYKDDDK (SEQ ID NO: 7) or YPYDVPDYA (SEQ ID NO: 8). In some embodiments, the epitope tag includes multiple arginine residues (e.g., RRRR, SEQ ID NO: 29). In some embodiments, the epitope tag includes multiple histidine residues (e.g., HHHHHH, SEQ ID NO: 30). In some embodiments, the epitope tag includes multiple histidine and arginine residues (e.g., HHHHHH-RRRR, SEQ ID NO: 31). d) Detectable label

[0071] In embodiments of the present disclosure, the polypeptide probe is detectably labeled. The labeled peptide can be used, for example, to better understand the mechanism of action and / or cellular location of the inhibitory peptide. Suitable labels that enable detection (e.g., provide a detectable signal or are detectable) are conventional and well known to those of skill in the art. Suitable detectable labels include, for example, radioactively active agents, fluorescent labels, and the like. Methods for conjugating such labels to proteins, or assays for detecting their presence and / or amount, are conventional and well known.

[0072] The polypeptide probe may further include one or more detectable labels. Suitable detectable labels that can be conjugated to the peptide are known in the art. In non-limiting examples, detectable labels include TMR, tetramethylrhodamine (e.g., TAMRA) or fluorescein isothiocyanate (FITC). In some embodiments, a linker is required to connect the label to the peptide. For example, conjugating FITC to a peptide requires a secondary linker (e.g., aminohexanoic acid). Thus, in some embodiments, the polypeptide further includes an aminohexanoic acid linker. 2. Exemplary polypeptide probe

[0073] According to various aspects of the present disclosure, the polypeptide probe may include the amino acid sequence RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 9). In other aspects, the polypeptide probe may include the amino acid sequence YPYDVPDYARRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 10), DYKDDDDKRRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 11) or HHHHHHRRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 12). In some aspects, the polypeptide probe may consist of the amino acid sequence YPYDVPDYARRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 10), DYKDDDDKRRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 11) or HHHHHHRRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 12) and a detectable label.

[0074] In a further aspect of the present disclosure, the polypeptide probe is selected from TAMRA-YPYDVPDYA-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 14), TAMRA-DYKDDDDK-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 15), TAMRA-HHHHHH-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 16), FITC-Ahx-YPYDVPDYA-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 17), FITC-Ahx-DYKDDDDK-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 18), FITC-Ahx-HHHHHH-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 19), FITC-Ahx-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 20), where TAMRA is tetramethylrhodamine, FITC is fluorescein isothiocyanate (FITC), and Ahx is an aminohexanoic acid linker.

[0075] In further embodiments, the polypeptide probe is selected from FITC-Ahx-HHHHHH-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 19), FITC-Ahx-YPYDVPDYA-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 17), and FITC-Ahx-DYKDDDDK-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 18). For example, in some embodiments, the polypeptide probe comprises FITC-Ahx-HHHHHH-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 19). In some embodiments, the polypeptide probe consists of FITC-Ahx-HHHHHH-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 19), and in any of these embodiments, FITC is fluorescein isothiocyanate (FITC) and Ahx is an aminohexanoic acid linker.

[0076] In various aspects, the polypeptide has an unexpectedly low detection threshold. As used herein, "detection threshold" refers to the concentration of a polypeptide that can be used and that can still record a recoverable signal in the presence of a detectable amount of a target (e.g., TTR fibrils). As further shown in Examples 2 and 16 below, the polypeptide probes used herein are very low detection threshold - probes as low as about 1 fM and have the ability to detect nanogram amounts of target (TTR fibrils). Thus, in some aspects, the lower limit of the detection concentration is 1 - 500 fM, 1 - 100 fM, 1 - 50 fM, 1 - 40 fM, 1 - 30 fM, 1 - 20 fM, 1 - 10 fM or 1 - 5 fM. In some aspects, the lower limit concentration of the polypeptide that can be used to detect TTR fibrils in a sample is about 1 fM, about 2 fM, about 3 fM, about 4 fM, about 5 fM, about 6 fM, about 7 fM, about 8 fM, about 9 fM or about 10 fM. In various aspects, the lower limit of the concentration of the polypeptide that can be used to detect TTR fibrils in a sample is about 1 fM.

[0077] In various embodiments, the polypeptide has an unexpectedly low EC50. As used herein, "EC50" refers to the concentration of a target that elicits a maximal half-maximal signal when detected by a standard concentration of a probe. In some embodiments, the polypeptide probe has an EC50 for a target of less than 1 μg. In some embodiments, the polypeptide probe has an EC50 for a target of about 1 - 100 ng. In some embodiments, the polypeptide probe has an EC50 for a target of about 10 - 50 ng, about 10 - 40 ng, about 10 - 30 ng, or about 20 - 30 ng. In some embodiments, the polypeptide probe may have an EC50 for a target of about 26.1 ng. Methods for determining EC50 for the probes provided herein are within the skill of the art and are further specifically described in the following examples (see FIGS. 19B and 16 below). 3. Variants and Modifications

[0078] In further embodiments, the polypeptide probe may further comprise one or more non-natural amino acids or modifications. In other embodiments, the polypeptide probe may not contain any non-natural amino acids or other modifications.

[0079] Amino acid substitutions. In some embodiments, amino acids other than those noted above in the consensus sequence are substituted. These amino acids may help protect the peptide against proteolysis or otherwise stabilize the peptide and / or otherwise contribute to desired pharmacodynamic properties. In some embodiments, non-natural amino acids enable the peptide to bind more tightly to the target because their side chains optimize hydrogen bonding and / or non-polar interactions therewith. Further, non-natural amino acids provide the opportunity to introduce detectable markers, such as strongly fluorescent markers that can be used, for example, to measure values such as inhibition constants. Also included are peptidomimetics, such as peptoids, beta amino acids, N-ethylated amino acids, and small molecule mimetics.

[0080] Non-natural amino acid substitution - In one embodiment, a non-natural amino acid substitutes for an amino acid in a sequence. More than 100 non-natural amino acids are commercially available. These include, for example, the non-natural amino acids listed in the following table.

Table A

Table B

Table C

Table D

Table E

Table F

[0081] In another embodiment, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) N-methylated residues are included in the peptide.

[0082]

[0083] For example, in some embodiments, the polypeptide probe may further include an N-methyl group. In other embodiments, the polypeptide probe does not include an N-methyl group. Various other types of active variants are encompassed.The polypeptide probes of the present disclosure can include, for example, L - amino acids, D - amino acids, non - natural amino acids, or combinations thereof. Active variants include molecules that contain various tags at the N - terminus or C - terminus of the peptide. For example, the polypeptides of the present disclosure can include, as tags, at their N - terminus and / or their C - terminus: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 lysine residues; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 arginine residues; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glutamic acid residues; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aspartic acid residues; combinations of these amino acid residues; or other polar tags apparent to those skilled in the art. In some embodiments, the tag can be provided within the peptide. Other active variants include mutations in the sequence of the polypeptide probe sequence that increase the affinity of the inhibitory peptide for the TTR molecule (oligomer, fiber, etc.).

[0084] In one embodiment of the present disclosure, the polypeptide probes described herein are isolated or purified using conventional techniques such as the methods described herein. "Isolated" means that it is separated from the components with which it is normally associated, for example, after the peptide probe has been synthesized Means being separated from the components present. An isolated polypeptide probe can be a cleavage product of a protein containing the polypeptide sequence. A "purified" polypeptide probe can be, for example, purer than 80%, 85%, 90%, 95%, 98% or 99%. If the polypeptide probes described herein contain a detectable label, the polypeptide portion of the probe can be synthesized and then conjugated to the detectable label. In some embodiments, the pharmaceutical composition can include polypeptide probes lacking a detectable label as long as at least one polypeptide probe in the composition contains a detectable label. In some embodiments, compositions are provided that include labeled polypeptide probes and, optionally, unlabeled polypeptide probes, with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the polypeptide probes being conjugated to a detectable label.

[0085] The polypeptide probes of the present disclosure can be synthesized by any of a variety of methods recognized in the art (e.g., chemically or by recombinant expression in a suitable host cell). To generate a sufficient amount of inhibitory peptide for use in the methods of the present disclosure, one of ordinary skill in the art can, for example, use conventional techniques to generate a nucleic acid (e.g., DNA) encoding the peptide and insert it into an expression vector, in which the sequence is under the control of expression control sequences, such as a promoter or enhancer, that can then direct the synthesis of the peptide. For example, one of ordinary skill in the art can (a) synthesize the DNA de novo with appropriate linkers for cloning it into the vector at its termini; (b) clone the entire DNA sequence into the vector; or (c) start with overlapping oligonucleotides, join them by conventional PCR-based gene synthesis methods, and insert the resulting DNA into the vector. Appropriate expression vectors (e.g., plasmid vectors, viral vectors including phages, artificial vectors, yeast vectors, eukaryotic vectors, etc.) will be apparent to those of ordinary skill in the art, as will methods for making the vectors, methods for inserting the sequence of interest, methods for expressing the protein encoded by the nucleic acid, and methods for isolating or purifying the expressed protein. C. Pharmaceutical Compositions

[0086] Any polypeptide probe disclosed herein can be formulated into a pharmaceutical composition. In some embodiments, the pharmaceutical composition can further comprise one or more pharmaceutically acceptable carriers, diluents, or excipients. Any of the pharmaceutical compositions used in the methods of the present invention can contain pharmaceutically acceptable carriers, excipients, or stabilizers in a lyophilized configuration or in the form of an aqueous solution.

[0087] The carrier in a pharmaceutical composition should be "acceptable" in the sense that it is compatible with the active ingredient of the composition, preferably capable of stabilizing the active ingredient, and not harmful to the subject being treated. For example, "pharmaceutically acceptable" can refer to the molecular entities and other ingredients of a composition that are physiologically tolerable and typically do not give rise to adverse reactions when administered to a mammal (e.g., a human). In some instances, a "pharmaceutically acceptable" carrier used in the pharmaceutical compositions disclosed herein can be one that has been approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in mammals, particularly humans.

[0088] Pharmaceutically acceptable carriers that include buffering agents are well known in the art and can include phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or nonionic surfactants. See, for example, Remington: The Science and Practice of Pharmacy 20 th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0089] The pharmaceutical compositions disclosed herein may also contain other ingredients, such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are non-toxic, preferably inert, to the recipient at the dosages and concentrations employed and include buffers, such as phosphates, citrates or other organic acids; antioxidants, such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as Tween®, Pluronic® or polyethylene glycol. III. Methods

[0090] In various aspects of the disclosure, the polypeptide probes provided herein can be used in methods for detecting transthyretin oligomers, fibrils or molecules in a subject in need thereof. The advantages of the methods herein are the ability of the polypeptide probes to detect aggregated forms of TTR (e.g., oligomers and fibrils), which allows for earlier detection of TTR-related conditions, and thus earlier diagnosis, along with more accurate risk determination of further TTR aggregation and improved therapeutic monitoring. Accordingly, methods for detecting transthyretin oligomers, fibrils or molecules in a subject, and methods of applying this detection for diagnosing, assessing risk, monitoring treatment, or treating individuals having or suspected of having a TTR-related disease or condition are provided herein below.

[0091] In one aspect, the present disclosure relates to a method for detecting oligomers, aggregates or fibrils of transthyretin in a sample, comprising: (a) contacting the sample with a polypeptide probe described herein; (b) binding the polypeptide probe to any oligomers, aggregates or fibrils of transthyretin in the sample; and (c) detecting a complex comprising the polypeptide and the oligomers, aggregates or fibrils of transthyretin, wherein the presence of the complex correlates with the presence of oligomers, aggregates or fibrils of transthyretin in the sample.

[0092] Another aspect of the present disclosure is a method for diagnosing a subject with a disease or condition (sometimes referred to herein as a TTR-mediated disease or condition) mediated by the presence of fibrillated or aggregated TTR. Such diseases or conditions include, for example, hereditary amyloidosis and wild-type ATTR amyloidosis. In some aspects, the method comprises: (a) detecting transthyretin oligomers, fibrils or molecules in a sample obtained from the subject using a polypeptide probe of the present disclosure according to the method provided herein; and (b) diagnosing the subject with a TTR-mediated disease or condition when transthyretin oligomers, fibrils or molecules are detected in the sample. In some aspects, the subject is diagnosed with a TTR-mediated disease or condition when the level of transthyretin oligomers, fibrils or molecules detected in the sample exceeds a threshold.

[0093] Another aspect of the disclosure is a method (e.g., a screening method) for determining whether a subject is at risk of TTR aggregation. In various aspects, the method comprises (a) detecting a transthyretin oligomer, fiber or molecule in a sample obtained from a subject using a polypeptide probe of the disclosure according to the methods provided herein, and (b) identifying the subject as being at risk of TTR aggregation if a transthyretin oligomer, fiber or molecule is detected in the sample. In some aspects, the subject is identified as being at risk of TTR aggregation if the level of transthyretin oligomers, fibers and / or molecules exceeds a threshold.

[0094] Another aspect of the disclosure is a method for monitoring the efficacy of a treatment administered to a subject for treating a TTR-mediated disease or condition. In some aspects, the method comprises (a) detecting a transthyretin oligomer, fiber or molecule in a first sample obtained from a subject using a polypeptide probe of the disclosure according to the methods provided herein, (b) administering a therapeutic agent to the subject, and (c) detecting a transthyretin oligomer, fiber or molecule in a second sample obtained from the subject after administration of the therapeutic agent, and (d) monitoring the efficacy of the therapeutic agent by comparing the level of detection of aggregated or fibrillar transthyretin in the first sample with the second sample. In various aspects, the therapeutic agent is determined to be effective if fewer transthyretin oligomers, aggregates and / or fibers are detected in the second sample compared to the first sample. In further aspects, the subject has been diagnosed with a TTR-mediated disease or condition using the methods provided herein (e.g., using a polypeptide probe). In some aspects, the method further comprises determining an effective amount of the therapeutic agent by monitoring the efficacy of different doses of the therapeutic agent. Suitable therapeutic agents that may be tested are described below.

[0095] Another aspect of the disclosure is a method of treating a subject with an effective amount of a therapeutic agent for a TTR-mediated disease or condition. In some aspects, the method includes determining an effective amount of a therapeutic agent for a TTR-mediated disease or condition in a subject using a method of determining the efficacy of the treatment provided herein, and then administering the effective amount of the therapeutic agent to the subject. In some aspects, the method includes diagnosing the subject with a TTR-mediated disease or condition as described herein, and then administering a therapeutic agent to the subject. In some aspects, the method includes determining that the subject is at risk of TTR aggregation according to the methods described herein, and then administering a therapeutic agent to the subject. Suitable therapeutic agents are described below.

[0096] In any of the preceding methods, the sample(s) can be obtained from a subject having or suspected of having transthyretin amyloidosis. In some embodiments, the sample(s) include a blood sample or a cerebrospinal fluid sample. In some embodiments, the sample(s) include a plasma sample. In various embodiments, the sample(s) include a tissue sample (e.g., a biopsy sample). In various embodiments, the tissue sample can include, but is not limited to, a fat biopsy, a nerve biopsy, or a heart biopsy. In various embodiments, the tissue sample includes heart tissue. For example, the sample can be a small heart biopsy. In some embodiments, the sample is obtained from a subject having the wild-type allele of the gene encoding transthyretin. In other embodiments, the sample is obtained from a subject having a variant allele of the transthyretin gene (e.g., a cardiothoracic-specific allele). For example, mutations in TTR, such as V122I, can lead to amyloid deposition in the heart and potentially heart failure, although some mutations including I68L and I84A can lead to neuropathy. Wild-type protein has also been shown to spontaneously aggregate and cause heart disease in cases of sporadic amyloidosis. In other embodiments, the sample is obtained from a subject having neuropathic ATTR with mutations including, among others, the early-onset V30M. In some embodiments, the sample(s) can be obtained from a carpal tunnel patient, a lifelong athlete, or a subject having or suspected of having HFpEF (heart failure with preserved ejection fraction). These conditions (e.g., carpal tunnel, sports or athletic activity, HFpEF) are conditions that can predispose a subject to TTR aggregation, where earlier detection and treatment are important. In various methods, the sample(s) can be obtained from a subject before the subject exhibits any signs or symptoms associated with TTR aggregation. In various methods, the sample(s) can be obtained from a subject after the subject begins to exhibit signs or symptoms associated with TTR aggregation.

[0097] In various aspects, suitable therapeutic agents used in previously described methods can be inhibitors of transthyretin expression and / or aggregation. In various aspects, the therapeutic agent includes an inhibitor of TTR expression. In some aspects, the therapeutic agent includes an inhibitor of TTR aggregation. Suitable inhibitors of TTR expression can include, for example, gene silencers that are agents that reduce or “silence” gene expression. Suitable gene silencers are known in the art and can include, for example, interfering RNAs (e.g., RNAi) and antisense oligonucleotides (ASO). Suitable inhibitors of TTR aggregation can include any agent (e.g., small molecule or antibody) that binds to TTR monomers or fibrils and stabilizes their conformation to prevent association and aggregation. In some aspects, the therapeutic agent includes tafamidis. In some aspects, the therapeutic agent includes an anti-TTR antibody or a gene silencer (e.g., siRNA or antisense oligonucleotide that targets the TTR gene or TTR mRNA).

[0098] In any of the above aspects, TTR-related disorders or diseases can include ATTR amyloidosis (e.g., wild-type ATTR amyloidosis or hereditary ATTR amyloidosis).

[0099] In any of the methods described herein, the subject can be a mammal. In any aspect described herein, the subject can be a human. *******

[0100] Although several embodiments have been described, those skilled in the art will recognize that various modifications, alternative configurations, and equivalents can be used without departing from the spirit of the disclosure. Further, some well-known processes and elements have not been described to avoid unnecessarily obscuring the present disclosure. Accordingly, this description should not be construed as limiting the scope of the disclosure.

[0101] Those skilled in the art will understand that the embodiments disclosed herein are taught by way of example and not limitation. Accordingly, the matters contained in this description or shown in the accompanying drawings should be construed as illustrative and not in a limiting sense. The following claims are intended, as a matter of language, to cover all the comprehensive and specific features described herein, as well as all statements of the scope of methods and assemblies, which may be said to fall within them.

Example

[0102] The following examples are included to demonstrate preferred embodiments of the present disclosure. The techniques disclosed in the following examples show techniques that have been found by the inventors to function well in the practice of the present disclosure, and thus it should be understood by those skilled in the art that they may be considered to constitute preferred modes for their implementation. However, those skilled in the art should understand that, in light of the present disclosure, many changes can be made in the disclosed specific embodiments without departing from the spirit and scope of the present disclosure and still obtain similar or analogous results. Introduction to Examples

[0103] ATTR amyloidosis is a highly underdiagnosed and lethal disease caused by the systemic deposition of amyloid fibrils composed of transthyretin (TTR when functional or ATTR when in its amyloidogenic form). ATTR deposition is induced by destabilization of the functional tetrameric form of TTR due to aging in wild-type ATTR (ATTRwt) amyloidosis or by mutation in variant ATTR (ATTRv) amyloidosis. The clinical picture of ATTRwt amyloidosis is relatively uniform, characterized mainly by cardiomyopathy and usually affects elderly men. ATTRv amyloidosis is often variable and unpredictable and can involve polyneuropathy, carpal tunnel syndrome, gastrointestinal and ocular lesions, and / or cardiomyopathy. Some studies have estimated the presence of pathogenic ATTRwt deposits in approximately 25% of the heart tissue of individuals over 80 years old. The most common mutations in the United States are ATTR-V30M, ATTR-T60A, and ATTR-V122I. Notably, the ATTR-V122I mutation is estimated to affect 3-4% of the African American population, making them more susceptible to the development of ATTRv amyloidosis and heart failure. Nevertheless, probably due to its heterogeneous clinical picture, this disease is underdiagnosed or misdiagnosed. These difficulties are amplified by the lack of robust tools that can detect the disease regardless of the patient phenotype.

[0104] The diagnostic process for ATTR amyloidosis is costly and complex, resulting in delayed patient diagnosis and ultimately a worse clinical prognosis due to a delayed start of treatment. After an individual is suspected of having amyloidosis (which can take several years to test for), the presence of amyloid should first be confirmed. Confirming amyloid deposition usually involves staining biopsy sections with Congo red dye. This dye has variable affinity and can bind to amyloid deposits from other precursor proteins, so it is not specific for ATTR. One of the biggest obstacles in the diagnostic process is the discrimination between ATTR amyloidosis and other forms of systemic amyloidosis, such as light chain amyloidosis. Thus, when the presence of amyloid is confirmed by Congo red, mass spectrometry or antibody-based assays are used to identify the protein forming the amyloid. However, mass spectrometry can require expensive equipment and highly trained individuals to evaluate the data, and the results can be inconclusive. Many commercially available antibodies are of low reliability in a diagnostic setting. In recent years, radiolabeled tracers, such as 99mTc-PYP, have become more common in detecting ATTR amyloid. Radiolabeled tracers are only appropriate for cardiac cases of ATTR amyloidosis, as they bind to calcification in the heart rather than amyloid. This technique requires the injection of radioactive reagents with potential health risks. Once the amyloid is confirmed to be ATTR, the clinician can genotype the patient to determine whether the patient has a mutation in the TTR protein. In summary, there is no single, robust, inexpensive, safe, and / or high-throughput tool that can diagnose ATTR amyloidosis. The development of biomarkers for early diagnosis would also reduce a very large burden not only on the resources of the healthcare system, but also on patients suffering from this disease and their families.

[0105] The novel biomarker indicates a potential tool for the diagnosis of ATTR amyloidosis. Blood biomarkers indicating heart injury, such as natriuretic peptides and cardiac troponin, are used to evaluate the severity and progression of ATTR amyloidosis, but are not specific for ATTR amyloidosis. Circulating transthyretin and its ligand retinol-binding protein 4 have also been evaluated as biomarkers for ATTR amyloidosis, but whether they can be used for diagnosis has not yet been determined. Recently, a peptide probe that binds to non-native ATTR species present in the plasma of patients with multiple neuropathy ATTRv has been developed in conjunction with an immunoassay that uses antibodies that specifically recognize these non-native oligomeric ATTR species only in the plasma of ATTR neuropathy patients. Additional tools are needed to specifically detect ATTR in patients regardless of their genotype or phenotype.

[0106] In the following examples, a first-generation transthyretin aggregation detection (TAD1) probe is described for the specific detection of ATTR fibers and aggregates in patient tissues. This probe shows high specificity for ATTR fibers in heart tissue, recognizes ATTR fibers in a conformation-dependent manner, and can be used as a tool to detect ATTR aggregates in plasma from patients with various genotypes and phenotypes. This novel tool can reveal the presence of aggregates in the blood and may represent a potential screening method for the specific detection of ATTR amyloidosis. (Example 1) Materials and methods for Examples 2 - 10

[0107] Dot blot of extracted fiber / patient lysate. The ability of the peptides to recognize TTR species was performed using dot blot analysis as described by Saelices et al (Saelices et al. Uncovering the mechanism of aggregation of human transthyretin. J. Biol. Chem. 2015. 27;290(48):28932-43). Briefly, 0.5 μg of fiber, lysate, or protein extracted from the hearts of ATTR patients was dotted onto a nitrocellulose membrane (0.2 μM, Bio-Rad). The membrane was blocked in 1×BSA / TBST for 30 minutes. After washing, the samples were then probed with the peptides in BSA / TBST for 1 hour. Fluorescence intensity was measured with an Azure Biosystems C600 imaging system via excitation of the membrane at 472 nm and read emission at 513 nm. Analysis of fluorescence intensity was performed using the software program GraphPad Prism.

[0108] Dot blot of blood samples. The ability of the peptides to recognize TTR species was performed using dot blot analysis as described by Saelices et al, 2015 (provided above) and tabulated in Figure 2. Briefly, a specified volume of patient or healthy blood was dotted onto a nitrocellulose membrane (0.2 uM, Bio-Rad). The membrane was blocked in 1×BSA / TBST for 30 minutes. After washing, the samples were then probed with the peptides in BSA / TBST for 1 hour. Fluorescence intensity was measured with an Azure Biosystems C600 imaging system via excitation of the membrane at 472 nm and read emission at 513 nm. Analysis of fluorescence intensity was performed using the software program GraphPad Prism.

[0109] Immunogold labeling / negative staining transmission electron microscopy. For immunogold labeling, 5 μM of the peptide was mixed overnight in binding buffer (20 mM Tris pH 7.6, 150 mM NaCl, 5 mM imidazole) with either 0.25 μM or 0.05 μM of Ni-NTA Nanogold beads (Nanoprobes). The next morning, the supernatant containing unbound nanogold beads was removed. 1 μg / ml of the fibrils extracted from wild-type ATTR patients was spiked into the pellet and the solution was loaded onto glow-discharged carbon-coated EM grids (Electron Microscopy Sciences). The solution was incubated for 2 minutes and then excess liquid was removed using filter paper. The grids were stained with 1% uranyl acetate for 1 minute. The grids were blotted again to remove excess dye and then visualized using a Tecnai Spirit electron microscope. The ability of the peptide to recognize TTR species was performed using dot blot analysis as described by Saelices et al, 2015. Briefly, 0.5 μg of fibrils, lysates or proteins extracted from ATTR patient hearts were dotted onto nitrocellulose membranes (0.2 μM, Bio-Rad). The membranes were blocked in l×BSA / TBST for 30 minutes to prevent non-specific binding of the probe. After washing, the samples were then probed with the peptide in BSA / TBST for 1 hour. Fluorescence intensity was measured with an Azure Biosystems C600 imaging system via excitation of the membrane at 472 nm and readout emission at 513 nm.

[0110] Peptide generation. The peptides were synthesized by LifeTein and sent for use in lyophilized form. The peptide probes used in Examples 2-10 have the sequence: "FITC"-"Ahx"-HHHHHH-RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT (SEQ ID NO: 12), where "FITC" is fluorescein isothiocyanate and "Ahx" is aminohexanoic acid. Before performing the experiments, the peptides were reconstituted in distilled water to a final concentration of 5 mM. The resuspended peptides were filtered through a 0.22 μm filter tube by spinning at 20,000 g for 5 minutes at 4 °C to remove any aggregates from the solution. (Example 2) The peptide probe selectively labels ATTR target fibrils

[0111] Dot blots were performed on ATTR fibrils extracted from 6 patients (3 with wild-type TTR genotype and 3 with TTR mutations), as well as the following controls: wild-type tetrameric TTR (recombinant (Rec) WT), tetrameric TTR with a mutation that stabilizes the tetramer (T119M) (recombinant TTR-T119M), recombinant monomeric TTR, serum amyloid protein (SAP, a protein reported to associate with ATTR amyloid fibrils), collagenase (an enzyme used to digest collagen when extracting ATTR fibrils from heart and brain lysates), and tissue extracted from Alzheimer's disease patients confirmed to contain amyloid and used as a negative control to show specificity for ATTR fibrils. Figure 3 shows how specific the tested probes (at nM concentrations) were for ATTR fibrils and not for native proteins or other fibrils.

[0112] To determine the lower limit of detection of the polypeptide probe, different amounts of I84S fibrils (e.g., 10 - 500 ng) were loaded onto nitrocellulose membranes and probed with 1 fM of the peptide. Figure 4 shows that the probe can detect 40 ng of fibrils loaded onto the membrane, which represents a significantly lower detection level than expected.

[0113] In another experiment, dot blotting was performed with crude heart lysates from the same patients (4 wild-type ATTR patients, second row; then 5 mutant ATTR patients, third row) using the same control samples as used in Figure 3. Figure 5 shows that our probe not only recognizes purified fibrils (e.g., Figure 3), but also fibrils within crude tissue preparations from both WT and mutant ATTR patients.

[0114] In another experiment, dot blotting was performed on 5 serum samples to determine whether the probe can recognize ATTR fibrils or aggregates within the serum. These samples included 1 non-amyloid control, 1 light chain (AL) amyloidosis patient, and 3 ATTR amyloidosis patients (2 wild-type TTR, 1 with a mutation). The results shown in Figure 6 demonstrate that the probe can detect fibrils or larger aggregates within the serum and that a reduction in signal can be detected after administration of treatment (e.g., tafamidis, the current treatment for ATTR amyloidosis), which implies that these probes have the potential to measure changes in pathogenic ATTR species during the duration of the treatment process. This data supports the surprising and unexpected use of these probes, that they can be used to measure disease progression and enable more effective therapeutics. (Example 3) The peptide probe binds to ATTR fibrils

[0115] The ability of the peptide probes to bind to ATTR fibers was tested using immunogold labeling as described in Example 1. Figure 7 shows a schematic overview of the experimental protocol used. Figures 8A and 8B show the labeling and binding of 5 μM of the peptide to ATTR wild-type fibers in the presence of 50 nM of Ni-NTA Nanogold (Figure 8A) or 250 nM of Ni-NTA Nanogold (Figure 8B). The labeled peptide localizes to the extracted ATTR fibers. In contrast, as shown in Figures 9A and 9B, no labeling was detected between the peptide and tau fibers. These results indicate that these peptides are specific for ATTR fibers but not for other types of amyloid. (Example 4) The peptide probe is specific for the conformation of ATTR fibers

[0116] To determine whether the peptide probe had a specific affinity for the TTR sequence or the conformation of TTR fibers (Figure 10), another experiment was conducted in which a detergent (SDS) was applied to the ATTR fibers prior to immunoblot analysis. We performed dot blotting of fibers extracted from ATTR amyloidosis patients and crude heart lysates from the same patients. We then subjected these samples to denaturing conditions by the addition of SDS and heating, and performed another dot blot probing for total transthyretin as well as the fibers. As shown in Figure 11, the application of the detergent abrogated binding to the pure fiber sample and significantly reduced binding to the heart lysate. This suggests that the peptide probe is structurally selective for ATTR fibers and shows little off-target binding to native-folded TTR or unfolded TTR peptides. Taken together, these data indicate that these peptide probes function in a conformation-dependent manner rather than in a sequence-dependent manner. (Example 5) The peptide probe can bind to ATTR aggregate species in plasma

[0117] Experiments were conducted to evaluate the specificity of the probe in serum samples versus plasma samples. Binding of the peptide to ATTR aggregates was evaluated using dot blot analysis as previously described (Saelices et al., J Biol Chem. 2015 Nov 27;290(48):28932-43). Briefly, serum or plasma was obtained from a cohort consisting of either pre-treatment ATTR amyloidosis patients, post-treatment ATTR amyloidosis patients, or healthy controls. 30 μL of each sample was dotted onto a nitrocellulose membrane (0.2 μm, Bio-Rad). The membrane was blocked for 30 minutes in 1× bovine serum albumin (BSA) in tris-buffered saline, 0.1% Tween-20 (TBST). After washing, the samples were probed with 15 μM TAD1 in 1× BSA / TBST for 1 hour. Fluorescence intensity of peptide binding was measured with an Azure Biosystems C600 imaging system via excitation of the membrane at 472 nm and emission read at 513 nm. Fluorescence intensity of binding was quantified via Image J and analyzed using Prism software. Results are shown for three technical replicates. FIG. 12 shows that the probe was able to successfully label plasma samples but not serum samples and can be used to diagnose ATTR amyloidosis in patients and monitor treatment response. (Example 6) The peptide probe is capable of binding to ATTR fibers in isolated tissue samples.

[0118] The binding of peptides to ATTR aggregates was evaluated using dot blot analysis as previously described by the inventors (Saelices et al., J Biol Chem. 2015 Nov 27;290(48):28932-43). Briefly, 0.5 μg of ex-vivo ATTR cardiac fibers, heart lysates, recombinant proteins, and control samples were dotted onto nitrocellulose membranes (0.2 μm, Bio-Rad). The membranes were blocked for 30 minutes in 1× bovine serum albumin (BSA) in tris-buffered saline, 0.1% Tween-20 (TBST). After washing, the samples were probed with TAD1 in 1× BSA / TBST for 1 hour. The fluorescence intensity of TAD1 binding was measured with an Azure Biosystems C600 imaging system via excitation of the membrane at 472 nm and readout emission at 513 nm. The fluorescence intensity of each dot was quantified via Image J and analyzed using Prism software. The results represent three technical replicates. Figures 13A and 13B show effective targeting of the probe to tissue samples, suggesting that the probe could be used as a diagnostic tool in small tissue samples or biopsies. (Example 7) Probe affinity for proteins in the native (folded) state

[0119] In various experiments, the affinity of the probe for folded proteins, oligomers or peptides was tested. Native gel electrophoresis was performed using the NativePAGE Novex Bis-Tris System (Invitrogen). 5 μg of ex-vivo ATTR cardiac fibers, 5 μg of recombinant protein or 20 μg of tissue lysate were mixed with NativePAGE 4× sample buffer according to the manufacturer's recommendations. 10 μL of each sample was loaded into the wells of a NativePAGE 4-12% Bis-Tris gel (Invitrogen). The gel was run at 150 V for approximately 2 hours at 4°C and transferred onto a 0.2 μm PVDF membrane (Millipore) using the Mini Trans-Blot cell system (BioRad) at 25 V for 1 hour. Proteins were immobilized on the membrane by incubation with 8% acetic acid. The membrane was then placed in 10% milk in TBST or 1× BSA for 1 hour to prevent non-specific binding. The membrane was washed three times consecutively in TBST for 5 minutes. The membrane was then incubated overnight with an anti-TTR antibody (1:1,000; Invitrogen) in 5% milk or with 5 μM peptide in 1× BSA / TBST for 1 hour. The membrane was then washed three times for 10 minutes. The fluorescence intensity of the peptide binding was measured as previously described. The membrane probed with the anti-TTR antibody was further incubated with a goat anti-rabbit secondary antibody (1:1,000; Invitrogen). The membrane was washed three times for 10 minutes and then incubated with an enhanced chemiluminescence reagent (Promega). The blot was imaged using an Azure Biosystems C600 imaging system. Figures 14A and 14B show that the probe bound successfully to high molecular weight (>10 kDa) folded species but did not bind to small oligomers, tetramers or monomers (all in folded states). (Example 8) Probe affinity for denatured (unfolded) state proteins

[0120] The same experiment as in Example 7 was repeated with the following changes. 5 μg of each sample (fiber, lysate, or recombinant protein) was dotted onto a nitrocellulose membrane (0.2 μm, Bio-Rad). The same samples were also added to beta-mercaptoethanol (a denaturing agent) and boiled at 95 °C for 10 minutes. These samples were dotted onto the second row of the membrane. The membrane was subjected to the same conditions as described for the native gel experiment. This was probed with either 5 μM of the peptide (in this case, the blocking and probing steps were carried out in BSA / 1×TBST) or an anti-transthyretin antibody at a concentration of 1:1,000 (in this case, the blocking and probing steps were completed in milk). The blot was imaged with an Azure Biosystems C600 imaging system. Figure 15 shows that the probe bound successfully to high molecular weight (>10 kDa) species but did not bind to small oligomers, tetramers, or monomers (in the unfolded state). Collectively, the data shown in Examples 7 and 8 (Figures 14A, 14B, and 15) indicate that the probe binds preferentially to high molecular weight species and does not bind to small oligomers, tetramers, or monomers, whether in the folded or unfolded state. (Example 9) Probe Validation in Patient Samples

[0121] Test the ability of the peptide probes to bind to ATTR fibrils in different patient samples. Plasma, serum, and tissue biopsy samples are obtained from patients with symptomatic ATTRwt (with cardiac phenotype), symptomatic ATTRv (with cardiac phenotype), symptomatic ATTRv (without cardiac phenotype), symptomatic AL (light chain amyloidosis characterized by aggregation of immunoglobulin light chain amyloid protein), symptomatic AA (another amyloidosis characterized by aggregation of serum amyloid A), or any other systemic amyloidosis, as well as from control asymptomatic non-carriers. Whenever possible, patient cohorts are matched for age and gender. Samples are processed and prepared as previously described and labeled with the peptide probes of the present disclosure. (Example 10) Monitor treatment outcomes

[0122] Monitor treatment outcomes using the polypeptide probes of the present disclosure. Specifically, tissue samples (plasma, serum, and tissue biopsies) are obtained from patients with symptomatic ATTRv (with cardiac phenotype), symptomatic ATTRv (without cardiac phenotype), and ATTRwt (with cardiac phenotype), as well as from control non-carriers, before and after treatment (with stabilizers and gene silencers). Treatment outcomes correlate with the detected levels of ATTR in each patient sample. (Example 11) Early detection with polypeptide probes

[0123] Perform additional experiments to measure the levels of ATTR in plasma and tissue biopsy samples obtained from pre-onset ATTRwt patients (with cardiac phenotypes), pre-onset ATTRv patients (with cardiac phenotypes), pre-onset ATTRv patients (without cardiac phenotypes), symptomatic ATTRwt patients (with cardiac phenotypes), symptomatic ATTRv patients (with cardiac phenotypes), symptomatic ATTRv patients (without cardiac phenotypes), and in patients considered at risk of developing ATTR amyloidosis, such as carpal tunnel patients, lumbar spinal stenosis patients, and patients with other orthopedic findings. This group may also include elderly lifelong high-intensity athletes and patients with heart failure with preserved ejection fraction (HFpEF). The levels of ATTR in samples from pre-onset patients correlate with symptom presentation and other markers of disease severity. (Example 12) Materials and methods for Examples 13 - 19.

[0124] Peptide design and synthesis. Peptide development was carried out by rational design starting with peptide inhibitors targeting two amyloidogenic segments of TTR (described in PCT / US17 / 40103, which is hereby incorporated by reference in its entirety). All sequences are listed in Table 1, as discussed below. Fluorescent and epitope modifications were added to the peptides to enable detection. The peptides were synthesized by LifeTein LLC and shipped for use in lyophilized form. Prior to performing the experiments, the peptides were reconstituted in distilled water to a final concentration of 1 mM. The resuspended peptides were filtered through 0.22 μm filter tubes by spinning at 20,000 × g for 5 minutes at 4°C to remove any possible aggregates from the solution.

[0125] Patients and tissue materials. Heart tissues from ATTR patients carrying wild-type TTR (n = 4) or TTR mutations (n = 7) included in the study are listed in Supplementary Table 2. Specimens from the left ventricle of either explanted or autopsy hearts were obtained from the laboratory of the late Dr. Merrill D. Benson at the University of Indiana. Serum and plasma samples from ATTR patients carrying wild-type TTR (n = 88) or TTR mutations (n = 34), as well as healthy controls (n = 32) and ATTRv carriers without cardiac symptoms (n = 16) are listed in Supplementary Table 3. Serum and plasma specimens were obtained from Dr. Ahmad Masri at Oregon Health and Science University, Dr. Wilson Tang and Dr. Mazen Hanna at the Cleveland Clinic, and the Dallas Heart Study at UTSW. Since all specimens were anonymized, the Office of the Human Research Protection Program granted an exemption from review by the Internal Review Board.

[0126] Extraction of amyloid fibrils from human heart tissue. Ex vivo fibrils were extracted from fresh frozen heart tissue as described by Nguyen, Afrin, et al bioRxiv, 2022.2006.2021.496949 (2022), which is hereby incorporated by reference in its entirety. Briefly, approximately 100 mg of frozen heart tissue per patient was thawed at room temperature and cut into small pieces with a scalpel. The finely chopped tissue was suspended in 400 μL of Tris-calcium buffer (20 mM Tris, 138 mM NaCl, 2 mM CaCl2, 0.1% NaN3, pH 8.0) and centrifuged at 3,100 × g for 5 minutes at 4°C. The pellet was washed and centrifuged three more times in Tris-calcium buffer. After washing, the pellet was resuspended in 375 μL of 5 mg / mL collagenase (Sigma Aldrich) in Tris-calcium buffer. This solution was incubated overnight at 37°C while shaking at 400 rpm. The resuspension was centrifuged at 3,100 × g for 30 minutes at 4°C, and the pellet was resuspended in 400 μL of Tris-ethylenediaminetetraacetic acid (EDTA) buffer (20 mM Tris, 140 mM NaCl, 10 mM EDTA, 0.1% NaN3, pH 8.0). The suspension was centrifuged at 3,100 × g for 5 minutes at 4°C, and the washing step with Tris-EDTA was repeated nine more times. After washing, the pellet was resuspended in 200 μL of ice-cold water supplemented with 5 mM EDTA and centrifuged at 3,100 × g for 5 minutes at 4°C. This step released the amyloid fibrils from the pellet, which were collected in the supernatant. EDTA assisted in solubilizing the fibrils. This extraction step was repeated five more times. Materials from various patients were handled and analyzed separately.

[0127] Preparation of fibril seeds. The extracted fibrils were treated with 1% sodium dodecyl sulfate, and the soluble fraction was discarded after centrifugation at 13,000 rpm for 5 minutes. This process was repeated twice, discarding the supernatant each time. The sample was washed three times with 10 mM sodium acetate (pH 7.5), 100 mM KCl and 1 mM EDTA, and then sonicated for a total of 10 minutes in 5-second on / 5-second off cycles. Protein content was measured using the Pierce Micro BCA Protein Assay Kit (Thermo Fisher Scientific).

[0128] Peptide thioflavin screening. The thioflavin T (ThT) fluorescence assay was used as an indirect measure of peptide binding to fibrils according to published protocols (Saelices, L. et al Proc Natl Acad Sci U S A 115, E6741-E6750 (2018) and Saelices, L. et al. J Biol Chem 294, 6130-6141 (2019)). Peptide binding to fibril seeds delays or inhibits the seeding of soluble TTR and the formation of amyloid fibrils, which is monitored by the fluorescence of ThT. 30 ng / mL of seeds were added to 200 μL of recombinant transthyretin at 0.5 mg / mL in a final volume of 5 μM ThT, 10 mM sodium acetate (pH 7.5), 100 mM KCl and 1 mM EDTA. The plate was incubated at 37 °C for 132 hours with shaking at 700 rpm. ThT fluorescence emission was measured at 482 nm using absorption at 440 nm in a FLUOstar Omega (BMG LabTech) microplate reader.

[0129] Preparation of patient-derived crude heart lysate. For the preparation of crude tissue lysate, 5 mg of patient heart tissue was suspended in 500 μL of Tris-calcium buffer containing protease inhibitor cocktail (Sigma Aldrich). The sample was homogenized using a biomasher (Polysciences) for 5 minutes. The sample was then placed in a bath sonicator and pulsed therein with an amplitude of 80 for 10 minutes, 5 seconds on and 5 seconds off. The protein concentration in the lysate was determined using the Pierce Micro BCA Protein Assay Kit (Thermo Scientific).

[0130] Fluorescent immunodot blotting of extracted fibers, crude heart lysate, and blood samples. Binding of TAD1 to ATTR species was evaluated using immunodot blot analysis as previously described by the inventors (Saelices, L. et al. J Biol Chem 290, 28932-28943 (2015)). Unless otherwise stated, 0.5 μg of ex-vivo ATTR cardiac fibers, heart lysate, recombinant protein, and control samples were dotted onto nitrocellulose membranes (0.2 μm, Bio-Rad). For assays using blood samples, unless otherwise specified, 30 μL of the sample was dotted onto the membrane. The membranes were blocked in 1× bovine serum albumin (BSA) in tris-buffered saline, 0.1% Tween-20 (TBST) for 30 minutes. After washing, the samples were probed with TAD1 in 1× BSA / TBST for 1 hour or overnight for blood samples. The fluorescence intensity of TAD1 binding was measured using an Azure Biosystems C600 imaging system via excitation of the membrane at 472 nm and emission reading at 513 nm. The TAD1 fluorescence intensity was quantified using ImageJ software. The signal was normalized against the highest fluorescence intensity on the membrane. For blood samples, the signal was normalized with respect to the intensity of ATTRwt fibers.

[0131] In vitro aggregation assay of TTR. The generation of recombinant transthyretin aggregates was performed as previously described (Saelices, L. et al. J Biol Chem 290, 28932-28943 (2015)). Briefly, tetrameric transthyretin with an N-terminal polyhistidine tag at 1 mg / mL was incubated at 37 °C for 4 days in 10 mM sodium acetate (pH 4.3), 100 mM KCl and 1 mM EDTA. At specified time points, 35 μL of the reaction was removed at each time point for anti-TTR and TAD1 immunodot blotting as described above. Additionally, 50 μL of the reaction was collected and spun at 13,000 rpm for 30 minutes to pellet the insoluble aggregates. The supernatant was removed and the pellet was resuspended in 50 μL of fresh aggregation buffer and the centrifugation step was repeated. After the second centrifugation, the supernatant was removed and the pellet was resuspended in 50 μL of 6 M guanidine hydrochloride. 2 μL of this mixture was added to 18 μL of guanidine hydrochloride prior to analysis. The polyhistidine tag in the insoluble fraction was probed with the SuperSignal West HisProbe Kit (ThermoFisher Scientific) according to the manufacturer's recommendations with the following modifications: instead of probing with a 1:5,000 HisProbe-HRP working solution, the membrane was probed with a 1:20,000 working solution.

[0132] Denaturing Western blot of extracted fibers and crude heart lysates. 1 μg of recombinant protein or 5 μg of ex-vivo ATTR cardiac fibers were boiled at 95 °C for 10 minutes. Samples were loaded onto three independent SurePAGE Bis-Tris 10×8 4–12% gels (Genscript) and run at 150 V for approximately 1 hour. One gel was processed for immunostaining using an anti-TTR polyclonal antibody, one gel was processed for detection of fluorescence upon binding to TAD1, and one gel was used as a control for Coomassie staining. Two gels were transferred onto nitrocellulose membranes at 25 V for 16 minutes using the Trans Blot Turbo System (BioRad). Membranes were placed in 10% milk in TBST or 1× BSA for 1 hour to prevent non-specific binding and washed three times consecutively in TBST for 5 minutes. One membrane was incubated overnight with an anti-TTR antibody (1:1,000; Genscript) in 5% milk, and the other membrane was incubated with 5 μM TAD1 in 1× BSA / TBST for 1 hour. The membranes were then washed three times for 10 minutes. Fluorescence intensity of TAD1 binding was measured as described above. Membranes probed with the anti-TTR antibody were further incubated with a goat anti-rabbit secondary antibody (1:1,000; Invitrogen). Membranes were washed three times for 10 minutes and then incubated with enhanced chemiluminescence reagent (Promega). Blots were imaged with an Azure Biosystems C600 imaging system. The third gel was stained with Coomassie using SimplyBlue SafeStain (Thermo Fisher Scientific). Gels were washed three times with distilled water for 5 minutes each to remove sodium dodecyl sulfate. This was then range-heated in distilled water for 1 minute 30 seconds and then shaken for 1 minute. This process was repeated two more times. Distilled water was then replaced with SimplyBlue SafeStain and range-heated for 30 seconds. Gels were incubated in the dye for 10 minutes and then destained in distilled water and imaged with an Azure Biosystems C600.

[0133] Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the extracted fibers and crude lysates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed using the NativePAGE Novex Bis-Tris System (Invitrogen). 5 μg of ex-vivo ATTR cardiac fibers, 5 μg of recombinant protein, or 20 μg of tissue lysate was mixed with NativePAGE 4× sample buffer according to the manufacturer's recommendations. 10 μL of each sample was loaded into the wells of three independent NativePAGE 4-12% Bis-Tris gels (Invitrogen). The gels were run at 150 V for approximately 2 hours at 4 °C and transferred onto a 0.2 μm PVDF membrane (Millipore) using the Mini Trans-Blot cell system (BioRad) at 25 V for 1 hour. The proteins were fixed to the membrane by incubation for 15 minutes with 8% acetic acid. The membrane was then subjected to the same staining protocol as previously described, with the following modifications: The membrane was incubated overnight with either a primary anti-TTR antibody (1:1,000; Genscript) or 5 μM of TAD1. For Coomassie staining, the gels were run using a dark blue cathode buffer as recommended by the manufacturer. After running the gels, the gels were placed in a fixing solution (40% methanol, 8% acetic acid), heated in a microwave for 45 seconds, and placed on an orbital shaker for 15 minutes. The solution was then decanted, and the gels were placed in a discoloring solution (8% acetic acid) until the desired background was obtained.

[0134] Filtration assay using patient plasma. 60 μL plasma samples from ATTR patients and healthy controls were subjected to filtration using 0.22 μM centrifugal filter tubes (Corning). The samples were spun at 1000 g at 4 °C at 10-second intervals until 20 μL of filtrate was present at the bottom of the tube. 20 μL of unfiltered plasma, i.e., plasma that had not passed through the filter (void), and plasma that had passed through the 0.22 μM filter (filtrate) were dotted onto nitrocellulose membranes as described above and probed with 10 μM of TAD1 and anti-TTR antibody.

[0135] Native gel shift assay using TAD1. 30 μg of ATTRwt patient plasma or healthy control plasma was incubated overnight with increasing concentrations of TAD1 (0, 12.5, 25, 50, 100 μM of TAD1). The samples were then subjected to gel electrophoresis under native conditions, and then Western blotting using an anti-TTR antibody was performed as described above. For quantification, the band patterns were classified into three groups (high molecular weight aggregates, oligomers, and tetramers) based on their associated molecular weights. These bands were quantified using ImageJ software (Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Nat Methods 9, 671-675 (2012)).

[0136] Immunogold labeling of ex vivo fibers. 5 μM of TAD1 was mixed with 50 nM or 250 nM of Ni-NTA Nanogold (Nanoprobes) in binding buffer (20 mM Tris pH 7.6, 150 mM NaCl) overnight at 4 °C. The next day, the nanogold beads were pelleted at the bottom of the reaction mixture. The supernatant containing unbound nanogold beads was removed. 1 μg / ml of fibers extracted from ATTRwt patients was spiked into the pellet, and the solution was loaded onto glow-discharge carbon-coated electron microscopy grids (Electron Microscopy Sciences, copper film 300 mesh). The solution was incubated for 2 minutes, and then excess liquid was removed using filter paper. The grids were stained with 1% uranyl acetate for 1 minute. The grids were blotted again to remove excess dye and then visualized using a Fei Tecnai Spirit transmission electron microscope (4K FEI CCD camera).

[0137] Statistical analysis. Statistical analysis of the TAD1 fluorescence, TTR aggregation, and ThT signals was performed using GraphPad Prism software. All samples were included in the analysis. All quantitative experiments were performed using three independent replicates and reported as the mean ± standard deviation of these replicates. Statistical significance between groups was compared using one-way ANOVA, where a cutoff of p < 0.05 was used to determine statistical significance between groups. Outliers in each dataset were identified and removed using the Grubbs test. (Example 13) Development of peptide probes.

[0138] The workflow for the design of the novel peptide probes is shown in Figure 16A. In previous studies, first-generation and second-generation transthyretin aggregation blocker peptides (TABs) that independently target the two amyloidogenic segments, β-strands F and H, of transthyretin were developed (see, e.g., Saelices, L. et al. Biol Chem 290, 28932-28943 (2015); Saelices, L. et al Proc Natl Acad Sci U S A 115, E6741-E6750 (2018); and Saelices, L. et al. J Biol Chem 294, 6130-6141 (2019), each of which is hereby incorporated by reference in its entirety). These peptides were designed to cap the ends of the fibers in order to inhibit protein aggregation and fiber elongation in vitro and in vivo. These peptides were found to inhibit a process known as amyloid seeding, in which mature ex-vivo ATTR fibers catalyze the formation of de novo amyloid fibers by templated addition of soluble recombinant transthyretin. In this study, the specific binding of these TAB peptide inhibitors was exploited for a different purpose, using them as detection probes. First, these peptides were optimized to generate third-generation TAB peptide inhibitors (TAB3) (Table 1).

Table 1

[0139] To screen and validate the TABs, their inhibitory effects on amyloid seeding were evaluated using the thioflavin T (ThT) assay and ex-vivo ATTRwt fibril seeds, prepared as previously described (see, for example, Saelices, L. et al Proc Natl Acad Sci U S A 115, E6741-E6750 (2018) and Saelices, L. et al. J Biol Chem 294, 6130-6141 (2019)). Since TAB3-12 (SEQ ID NO: 9) was found to be the most potent inhibitor (Figure 16B, blue), this peptide was selected for further modification. TAB3-12 was fused to an N-terminal epitope and a fluorescent tag to generate the first-generation transthyretin aggregation detection factor (TAD). Binding of TAD to fibrils was screened using an amyloid seeding inhibition assay as an agent, as performed for the peptide inhibitors, and it was found that TAD1 (containing a polyhistidine tag) did not induce fibril formation but instead completely inhibited seeding (Figure 16C). The sequences of all three TAD detection probes (TAD1, TAD2, and TAD3) are provided in Table 2 below. In the table, TAB3-12 (RRRRHVAHPFVEFTEGGGSTERRRRSYVTNPTSYAVT, SEQ ID NO: 9) is underlined in each probe.

Table 2

[0140] It was also confirmed that TAD1 did not form fibrils by itself and that TAD2 and TAD3 did not completely inhibit amyloid seeding (Figure 16C). Therefore, TAD1 (SEQ ID NO: 12) was selected for further study. (Example 14) Dot blotting as a simple experimental platform for the analysis of patient samples.

[0141] As described in previous examples (e.g., Examples 2-10), dot blotting was selected as the experimental platform to measure the direct binding of TAD1 to different samples (Figure 17), because dot blotting is technically and conceptually simple, does not favor sample types, and provides an unambiguous readout of the direct binding between two molecules. Briefly, the research samples were loaded onto a nitrocellulose membrane, then the membrane was incubated with TAD1, the membrane was washed to remove excess probe, and fluorescence was used to visualize the TAD1 binding to the research samples. Stronger dot intensity indicates more TAD1 binding to the sample. The relative intensity can then be quantified to indicate relative TAD1 binding. (Example 15) TAD1 binds to ATTR in patient-derived ATTR fibers and tissue lysates.

[0142] In addition to the experiments and data described in Examples 2-10, particularly Example 3, the ability of TAD1 to bind to ATTR fibers present in ATTR fibers purified from ATTR amyloidosis patients and ATTR heart lysates was tested. Fibers were extracted from fresh frozen heart tissue from 9 patients, 4 wild-type (ATTRwt) and 5 hereditary variants (ATTRv), as previously performed using the methods described in Nguyen, Afrin et al bioRxiv, 2022.2006.2021.496949 (2022) and the above paragraph

[0126] . Table 3 below provides a list of all ATTR heart tissue samples included in the study.

Table 3

[0143] 5 μg of fibers were loaded onto a nitrocellulose membrane along with controls and incubated with TAD1. The controls included recombinant tetrameric TTR, recombinant TTR with the T119M mutation, recombinant monomeric TTR variant (MTTR), TAB3-12, serum amyloid protein (SAP, which can associate with ATTR fibers), and collagenase (used in the fiber extraction process). To test the specificity of TAD1 for ATTR fibers, not only protein in a fibrillar conformation, but also recombinant tau fibers, and brain lysates from Alzheimer's disease (AD) patients were used as controls. It was found that TAD1 can be used for the detection of purified ATTR fibers regardless of the genotype of the ATTR patient (Figure 18A). Similarly, 5 μg of heart lysate from the same patients as in Figure 18A was loaded onto the membrane along with the same controls and incubated with 5 μM TAD1 (Figure 18B). Using this technique, ATTR species present in crude tissue lysates obtained from these patients were detected (Figure 18B). In none of the experiments was there detectable TAD1 binding to the control samples (Figures 18A - 18B). These experiments demonstrated that TAD1 can be used for the detection of patient-derived ATTR fibers and ATTR fibers within lysates in a specific manner, thereby validating the probe design strategy. (Example 16) TAD1 binds to ATTR fibers with high sensitivity and accuracy.

[0144] Next, the sensitivity and precision of the TAD1 dot blotting system were evaluated. To determine TAD1 sensitivity, different amounts of ATTRwt fibrils were titrated on membranes incubated with 5 μM TAD1. TAD1 was found to exhibit high sensitivity, recognizing approximately 20 ng of purified ATTRwt fibrils with a calculated EC50 of 26.1 ng (Figure 19B) (see also Figure 4 and Example 3). To evaluate the precision of the experimental setup and the variability of the fluorescence readout, three different amounts of ATTRwt fibrils (1.5 ng, 3 ng, and 6 ng) were loaded onto membranes and probed with 5 μM TAD1 (Figure 19C). Quantification of the fluorescence signal indicated that TAD1 could discriminate between small variations in the amount of fibrils with statistical significance (Figure 19D). These results demonstrate that the probe and experimental setup exhibit high sensitivity and precision, further supporting the data presented in Example 3 above. (Example 17) TAD1 reveals a novel blood biomarker in cardiac ATTR amyloidosis patients.

[0145] Transthyretin can adopt a non-native conformation in the blood of patients with neuropathic ATTRv amyloidosis, which may indicate an early stage of protein aggregation. Since TAD1 shows high sensitivity to ATTR fibrils and not to other forms of transthyretin, it was hypothesized that TAD1 could detect the presence of these non-native ATTR species (or different species) in the blood. Accordingly, serum and plasma samples from both ATTRwt patients and ATTRv amyloidosis patients (Table 4) were tested for the presence of TAD1-positive species. [Table 4-1] [Table 4-2]

[0146] First, pilot dot blotting assays of two ATTR samples and controls were performed by probing with 5 μM TAD1. Both serum and plasma were found to contain ATTR species detected by TAD1 (Figure 20A). Next, a small cohort of patient samples was analyzed to investigate which sample type (serum or plasma) might provide a better readout of the presence of these species (Figure 20B). In serum, quantification of the relative fluorescence intensity of TAD1 did not reveal a clear difference in binding when treated patients, untreated patients, and negative controls were compared (Figure 20B). In contrast, analysis of plasma samples revealed a significant difference between the negative control and pre-treatment ATTR patients, including both ATTRwt and ATTRv patients (Figure 20C). Regardless of treatment type, a significant difference was also found in the TAD1 signal between pre- and post-treatment (Figure 20C). These experiments indicated that serum does not contain TAD1-positive species detectable under these conditions, and thus further studies were performed using plasma samples.

[0147] Next, the pilot study data were extended to test TAD1 binding to species in plasma of a larger cohort of samples that included healthy sex- and age-matched controls, pre-treatment and post-treatment ATTR amyloidosis patients, pre-symptomatic ATTRv carriers, and immunoglobulin light chain amyloidosis (AL amyloidosis) patients (see Table 4 above). The TAD1 signal was found to be significantly higher in ATTR patients (pre-treatment) compared to healthy controls (Figure 21A). A significant difference in the TAD1 signal was also observed between pre-treatment and post-treatment ATTR patients (Figure 21A). Furthermore, the TAD1 signal in ATTRv carriers was detected before cardiac findings, indicating that TAD1 can be used as a tool for the early detection of these unique ATTR species in plasma (Figure 21A). No detectable TAD1 signal was present in plasma from AL amyloidosis patients, further demonstrating the specificity of TAD1 for ATTR (Figure 21B). These results validate the previous data presented in Examples 2-10 and suggest that ATTR plasma contains unique biomarkers that decrease in response to treatment and appear in the blood before symptoms. This biomarker may represent a powerful tool for early detection and monitoring of treatment response. (Example 18) TAD1 binds to recombinant proteins, ATTR fiber extracts, and large species from plasma.

[0148] It was unclear which molecular properties of the detected ATTR species were present in patient samples. Since TAD1 binds to ATTR fibers with high affinity, it was hypothesized that the detected species in patient samples could be high molecular weight ATTR aggregates. This hypothesis was tested using three types of samples: recombinant TTR aggregates, purified ex-vivo ATTR fibers, and plasma samples from ATTR patients (Figures 22A-22D and 24A-24B).

[0149] First, the binding of TAD1 to recombinant TTR aggregates made under acidic pH using wild-type transthyretin was evaluated as previously described (see, for example, Saelices, L. et al. J Biol Chem 290, 28932-28943 (2015)). In this standard procedure, the native tetrameric structure of transthyretin is dissociated, aggregation is promoted in vitro by lowering the pH of the sample to 4.3, and aggregation is monitored by measuring the absorbance at 400 nm. After performing this assay, aggregation can be visualized by dot blotting using an antibody that recognizes the polyhistidine tag of recombinant transthyretin (Figure 22A). The total amount of transthyretin appears not to change in the sample (Figure 22A, upper panel), while the TAD1-positive species increase over time (Figure 22A, middle panel). The TAD1 signal correlates with the increase in insoluble aggregates collected by centrifugation (Figure 22A, lower panel), suggesting that TAD1 binds to recombinant aggregates in a conformation-dependent manner.

[0150] To characterize the size of the TAD1-positive species in the purified fibrils, Western blotting was performed under non-denaturing and denaturing conditions (Figure 22B and Figures 23A and 23B). Under non-denaturing conditions, TAD1 was observed to bind to ATTR fibrils and recombinant transthyretin aggregates that do not flow through the gel. These aggregates correspond to molecular weights higher than 1048 kDa (Figure 22B, Figure 23A). When the same samples are subjected to denaturing conditions, TAD1 loses its ability to bind to aggregates or fragments resulting from denatured ATTR fibrils (Figure 23B), providing additional evidence that the recognition of ATTR species by TAD1 is conformation-dependent.

[0151] To characterize the binding of TAD1 to ATTR species in plasma, two complementary assays were performed. First, ATTR patient and healthy plasma were filtered using a 0.22 μM pore filter and evaluated by dot blotting to measure TAD1 binding to species in the filtrate and void (Figure 22C). TAD1 binds to aggregated high molecular weight TTR in plasma that cannot pass through the filter (Figure 22D). In the second assay, a non-denaturing protein-protein band shift experiment was performed that enables visualization of changes in the electrophoretic behavior of a protein (soluble or aggregated) upon binding to a second protein (Figure 24A). Briefly, ATTRwt patient plasma or negative control plasma was incubated with increasing concentrations of TAD1, electrophoresed on a non-denaturing gel, and probed with an anti-TTR antibody. The first interesting observation from this experiment was the presence of oligomeric ATTR species in ATTRwt plasma that were not present in the negative control (Figures 24A - 24C). Upon binding to TAD1, these oligomers as well as tetrameric soluble transthyretin disappeared in a concentration-dependent manner. In contrast, high molecular weight species that did not flow through the gel accumulated (Figures 24A - 24B). An increase in tetrameric soluble transthyretin in healthy plasma with increasing concentration of TAD1 was also observed (Figures 24A, 24D). Together, these results indicate that TAD1 recognizes aggregated ATTR species with high molecular weight in a conformation-dependent manner. (Example 19) TAD1 binds to ATTR via a unique mechanism.

[0152] Finally, we evaluated where and how TAD1 binds to ATTR fibrils. First, we tested whether the interaction of TAD1 with the ATTR species is electrostatic, as 18 out of 43 residues of TAD1 are charged amino acids under these experimental conditions. To test this hypothesis, TAD1 binding was assayed under multiple pHs (Figure 25A) and salt concentrations (Figure 25B). None of these changes were found to significantly affect the binding of TAD1 to purified ATTRwt fibrils, suggesting that the interaction could be hydrophobic. Next, in further experiments, we tested where on the purified ATTRwt fibrils the TAD1 interaction occurs. Utilizing the polyhistidine tag present in TAD1, this peptide was coated with nickel nitrilotriacetic acid nanogold beads upon binding to the purified ATTR fibrils (Figure 26A). Nanogold particles were observed to decorate the ATTR fibrils, mainly at the tips at low concentrations and on the surface of the fibrils at higher concentrations (Figure 26B). Tau fibrils were used as a negative control (Figure 26C). The high sensitivity binding of nanogold particles to the tips of the fibrils is consistent with our structure-based peptide design pipeline (Figures 16A - 16C). These experiments suggest that TAD1 binds to the ATTR species, likely through hydrophobic interactions with the tips and surface of the fibrils. Discussion of Examples

[0153] ATTR amyloidosis is a lethal disease that is likely to be underdiagnosed due to its complex diagnostic process. In recent years, there have been notable advancements in the development of treatment options for ATTR amyloidosis that are effective in halting disease progression when administered at an early stage. Therefore, improvement of the diagnostic process is important to enable early treatment and thus reduce the burden on patients. Herein, we designed peptides for the detection of cardiac ATTR fibrils and aggregates in cardiac tissue and plasma using the structure of the aggregation-driving segment of ATTR (Figures 16A - 16C). This peptide robustly detects ATTR fibrils purified from the heart and ATTR fibrils within cardiac lysates with high sensitivity, specificity, and accuracy (Figures 18A, 18B, 19A, 19B, 19C, and 19D). The data presented above also reveals novel biomarkers in plasma consisting of high molecular weight ATTR species (Figures 21A, 21B, 22A, 22B, 22C, 22D, 24A, and 24B).

[0154] This structure-based peptide detects novel plasma biomarkers in patients with cardiac ATTR amyloidosis. TAD1 also detects a unique ATTR species present in the blood of patients with ATTR amyloidosis that was not observed in either age-matched healthy controls or patients with other forms of systemic amyloidosis, not only in ATTR fibrils (Figures 21A and 21B). These species are high molecular weight aggregates that can accumulate in the blood prior to the onset of an individual's symptoms, as suggested by our results (Figures 21A, 21B, 22A, 22B, 22C, and 22D). When combined with previous results, these examples demonstrate that multiple types of non-native transthyretin species, including monomeric misfolded states and high molecular weight aggregates, can be present in the blood of patients with ATTR amyloidosis (Figures 21A, 21B, 22A, 22B, 22C, and 22D). Presumably, the decrease in soluble transthyretin observed in patient sera is associated with the formation of these insoluble species that can be detected in plasma. Our studies establish a novel plasma biomarker for cardiac ATTR amyloidosis and shed light on the fundamental biology of ATTR amyloid formation in the blood.

[0155] Current blood biomarkers for ATTR amyloidosis have limitations that the disclosed polypeptide probes address. Biomarkers, such as natriuretic peptides and cardiac troponin, are not specific for diagnosing cardiac ATTR amyloidosis and diagnose common cardiac injury. The reliability and specificity of other biomarkers, such as transthyretin and retinol-binding protein 4, are still unknown. The group is designing novel peptides and antibodies that use segments of TTR to bind to non-native TTR (NNTTR) in the plasma of neuropathy patients but have limited efficacy in patients with purely cardiac or mixed phenotypes. Another major challenge in using blood biomarkers for ATTR amyloidosis is to distinguish ATTR amyloidosis from other forms of systemic amyloidosis, such as immunoglobulin light chain amyloidosis (AL amyloidosis). The disclosed polypeptide probes robustly detect ATTR species in the plasma of cardiac ATTR amyloidosis patients, regardless of genotype or phenotype, and distinguish between ATTR amyloidosis and AL amyloidosis (Figure 21B). This strategy depends on the structure of ATTR fibrils to increase specificity, similar to that implemented by other groups for the development of binding factors for other amyloid fibrils. Structure-based diagnostics are a potential means to simplify the diagnostic process for cardiac ATTR amyloidosis and detect the structural shift between native TTR and its mature amyloidogenic conformation.

[0156] TAD1 can potentially be used for the detection of cardiac ATTR amyloidosis before the onset of an individual's symptoms (Figures 21A, 21B). Detection of ATTR species in blood ATTRv carriers without clinical findings of the disease suggests that transthyretin aggregation begins in the blood (Figures 21A, 21B, 22A, 22B, 22C, and 22D). As shown in patients with neuropathy, it has also been shown that misfolding of TTR begins in the blood. Early detection of TTR misfolding and ATTR aggregation in the blood may enable thorough monitoring of disease progression, thereby providing information about the need for early treatment. The correlation between TTR misfolding and ATTR aggregation in the blood, and the onset of the clinical phenotype, need to be established using conventional diagnostic methods to justify treatment.

[0157] Furthermore, TAD1 can potentially be used to monitor treatment response or to optimize the effective dose of a therapeutic agent. TAD1 fluorescence intensity decreases in the treatment group compared to the untreated group (Figures 21A and 21B). This result suggests that therapeutic agents designed to kinetically stabilize transthyretin reduce high molecular weight aggregates in the blood. Similar results have been observed when a small group of patients were tested with gene silencers, indicating that these treatments can also result in a reduction of ATTR aggregates in the blood. Studies detecting TTR misfolding in neuropathy patients after treatment with stabilizers, gene silencers, and liver transplantation also show a decrease in the presence of these misfolded species. Thus, these examples describe a new biomarker for cardiac ATTR amyloidosis that can be utilized to monitor treatment response or optimize treatment regimens.

[0158] In summary, the structure of ATTR fibers is used herein to design novel peptides for the detection of ATTR fibers and aggregates in patients with cardiac ATTR amyloidosis. This peptide has revealed a novel plasma biomarker consisting of high molecular weight aggregated transthyretin. This peptide has shown that it can detect these aggregates in the blood of ATTRv carriers before symptoms appear and can potentially be used for early detection. The observed reduction in signal in treated patients further indicates that this biomarker can be used to monitor treatment response. Finally, this peptide can be used for the study of the biology and pathogenesis of ATTR amyloidosis and can lead to the identification of new targets for therapeutic development. *******

[0159] The following table lists the sequences provided in the present application.

Table 5-1

Table 5-2

Table 5-3

Claims

1. A polypeptide probe comprising a first peptide containing the sequence HVAHPFVEFTE (SEQ ID NO: 1) and a second peptide containing the sequence SYVTNPTSYAVT (SEQ ID NO: 2), wherein the first and second peptides are covalently linked via a linker peptide, and the polypeptide probe further comprises a detectable label.

2. The polypeptide probe according to claim 1, wherein the first peptide and the second peptide are simultaneously bound to two different chains of transthyretin fibers or aggregates.

3. The polypeptide probe according to claim 2, wherein the two different chains of the transthyretin fiber or aggregate are an "F" chain and an "H" chain.

4. The polypeptide probe according to claim 1, wherein the linker peptide comprises the sequence GGGSTE (SEQ ID NO: 3), EAAAK (SEQ ID NO: 4), PAPAP (SEQ ID NO: 5), or GGGGGG (SEQ ID NO: 6).

5. The polypeptide probe according to claim 1, wherein the polypeptide further comprises an epitope tag that facilitates the dissolution, manipulation, and / or purification of the polypeptide, and / or the detectable label is covalently linked to the N-terminus of the first peptide or the C-terminus of the second peptide, optionally via a linker containing aminohexanoic acid (Ahx).

6. The polypeptide probe according to claim 1, wherein the polypeptide comprises the amino acid sequence RRRRRHVAHPFVEFTEGGGSTERRRRRRSSYVTNPTSYAVT (SEQ ID NO: 9), YPYDVPDYARRRRRRHVAHPFVEFTEGGGSTERRRRRRSSYVTNPTSYAVT (SEQ ID NO: 10), DYKDDDDK-RRRRRHVAHPFVEFTEGGGSTERRRRRRSSYVTNPTSYAVT (SEQ ID NO: 11), or HHHHHHRRRRRHVAHPFVEFTEGGGSTERRRRRRSSYVTNPTSYAVT (SEQ ID NO: 12).

7. The polypeptide probes are FITC-Ahx-HHHHHH-RRRRRHVAHPFVEFTEGGGSTERRRRRRSSYVTNPTSYAVT (SEQ ID NO: 19), FITC-Ahx-YPYDVPDYA-RRRRRHVAHPFVEFTEGGGSTERRRRRRSSYVTNPTSYAVT (SEQ ID NO: 17), FITC-Ahx-DYKDDDDK-RRRRRHVAHPFVEFTEGGGSTERRRRRRSSYVTNPTSYAVT (SEQ ID NO: 18), TAMRA-YPYDVPDYA-RRRRRHVAHPFVEFTEGGGSTERRRRRRSSYVTNPTSYAVT (SEQ ID NO: 14), TAMRA- A polypeptide probe according to claim 1, selected from the group consisting of DYKDDDDK-RRRRRHVAHPFVEFTEGGGSTERRRRRSYVTNPTSYAVT (SEQ ID NO: 15), TAMRA-HHHHHH-RRRRRHVAHPFVEFTEGGGSTERRRRRSYVTNPTSYAVT (SEQ ID NO: 16), and FITC-Ahx-RRRRRHVAHPFVEFTEGGGSTERRRRRSYVTNPTSYAVT (SEQ ID NO: 20), wherein TAMRA is tetramethylrhodamine, FITC is fluorescein isothiocyanate (FITC), and Ahx is an aminohexanoic acid linker.

8. A pharmaceutical composition comprising a polypeptide probe according to claim 1 and a pharmaceutically appropriate carrier or excipient.

9. A method for detecting transthyretin oligomers, aggregates, or fibers in a sample, comprising: (a) contacting the sample with a polypeptide probe according to claim 1; (b) binding the polypeptide probe to any transthyretin oligomers, aggregates, or fibers in the sample; and (c) detecting a complex comprising the polypeptide probe and transthyretin oligomers, aggregates, or fibers, wherein the presence of the complex correlates with the presence of transthyretin oligomers, aggregates, or fibers in the sample.

10. The aforementioned sample, (a) Obtained from subjects who have or are suspected of having transthyretin amyloidosis, who have the wild-type allele of the gene encoding transthyretin and / or the variant allele of the gene encoding transthyretin, (b) including blood samples, tissue samples, plasma samples or cerebrospinal fluid samples, and / or (c) including transthyretin-expressing tissue obtained from cardiac biopsy, fat biopsy, nerve biopsy, gastrointestinal biopsy and / or salivary gland biopsy, The method according to claim 9.

11. A method for obtaining oligomers, aggregates, or fibers of transthyretin in a sample as an indicator of whether a subject is at risk of TTR aggregation and / or has a TTR-related disorder or disease, comprising the step of detecting the oligomers, aggregates, or fibers of transthyretin in a sample obtained from the subject according to the method of claim 9, wherein if the oligomers, aggregates, or fibers of transthyretin are detected in the sample and exceed a threshold, it is indicated that the subject is at risk of TTR aggregation and / or has a TTR-related disorder or disease.

12. A method for obtaining oligomers, aggregates, or fibers of transthyretin in first and second samples as an indicator of the effectiveness of a therapeutic agent administered to a subject to treat a TTR-related disorder or disease, comprising: (a) detecting oligomers, aggregates, or fibers of transthyretin in a first sample obtained from a subject before the therapeutic agent is administered to the subject, according to the method of claim 9; and (b) detecting oligomers, aggregates, or fibers of transthyretin in a second sample obtained from the sample after the therapeutic agent has been administered to the subject, wherein the therapeutic agent is determined to be effective if fewer oligomers, aggregates, and / or fibers of transthyretin are detected in the second sample compared to the first sample.

13. A composition for treating a subject with a TTR-related disorder or disease, wherein the composition comprises a therapeutic agent, and the subject has been determined to be at risk of TTR aggregation and / or has been shown to have a TTR-related disorder or disease in accordance with claim 11.

14. A composition for treating a subject with respect to a TTR-related disorder or disease, wherein the composition comprises a therapeutic agent, and the effective amount of the therapeutic agent is determined according to the method described in Claim 12.

15. (a) The TTR-related disorder or disease includes ATTR amyloidosis, (b) The subject has or is suspected to have a state or characteristics that make the subject susceptible to TTR aggregation, (c) The subject is elderly, has carpal tunnel syndrome, is an athlete, has heart failure with preserved ejection fraction (HFpEF), has a mutation in the TTR gene, or any combination thereof, and / or (d) The subject has or is suspected of having transthyretin amyloidosis, has a wild-type allele of the gene encoding transthyretin, and / or has a variant allele of the gene encoding transthyretin, A method according to claim 11 or 12, or the composition according to claim 13 or 14.

16. The therapeutic agent is (a) Inhibitors of transthyretin expression and / or aggregation, (b) Small molecules, gene silencers or antibodies, and / or (c) Tafamijis A method according to claim 11 or 12, or a composition according to claim 13 or 14, comprising:

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