Diagnostic methods for amyloidosis and related disorders

Abstract

The present invention provides novel non-invasive methods for detecting misfolded transthyretin (TTR) proteins, and for diagnosing and monitoring amyloidosis and related disorders.

Description

Attorney Dkt No.: TSRI 2240.1PC Diagnostic Methods for Amyloidosis and Related Disorders CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The subject patent application claims the benefit of priority to US Provisional Patent Application No.63 / 703,279 (filed October 4, 2024; now pending). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes. STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under TR004407 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0003] This application includes by incorporation of reference a sequence listing in the XML format, 2240_1PC_Sequence Listing, which was created on September 16, 2025 and contains 12 KB in content. BACKGROUND

[0004] Amyloid transthyretin (ATTR) amyloidosis is a type of amyloidosis, a life- threatening disease in which buildup of abnormal proteins, in particular of misfolded transthyretin (TTR), called amyloid, occurs in the body’s organs and peripheral nerves leading to major health problems. These include cardiomyopathy, difficulty walking unassisted, ocular disease and polyneuropathy. The abnormal protein can be caused either by well-known hereditary mutations or by destabilization and aggregation of normal wildtype TTR, presumably due to aging. While ATTR amyloidosis is still considered a rare disease, there is growing evidence that the disease is largely underdiagnosed.

[0005] Clinical indications of ATTR amyloidosis include very common symptoms such as fatigue, heart problems, shortness of breath and gastrointestinal (GI) distress, which makes the disease difficult to recognize by its symptoms alone. Due to a lack of molecular diagnostic tests, ATTR amyloidosis is often confused with other conditions and it may take years until a patient is finally diagnosed with ATTR amyloidosis, if at all. Most patients areonly diagnosed when they already have so much amyloid that they have heart failure and are evaluated at specialized amyloidosis centers. In these settings, specialists can rule out or rule in ATTR diagnosis based on experience, tissue biopsies, and echocardiograms. In research settings, cardiac MRI or PET scans with radiotracers are used to confirm diagnosis. Unfortunately, these methods come with significant drawbacks such as high expense, very limited availability in a few specialized research centers and potential risks such as exposure to radiation as well as the need for specifically trained specialists to interpret the results.

[0006] There is a strong and urgent need for better means for diagnosis and monitoring of ATTR amyloidosis. The present invention addresses this and other unmet needs in the art. SUMMARY

[0007] In one aspect, the present invention provides methods for detecting the presence and / or quantifying degree of misfolding of proteins in a biological sample. The methods involve (a) generating at least one peptide from one or more misfolded proteins present in the biological sample, with different molecules of the at least one peptide containing one or more amino acid residues in both solvent accessible and solvent excluded states, (b) quantifying solvent accessibility and solvent exclusion for each of the one or more amino acid residues, and (c) calculating the ratios of solvent accessibility to solvent exclusion for each of the one or more amino acid residues. A decrease of the ratios for the one or more amino acid residues, relative to the ratios for the same amino acid residues in the same but correctly folded control proteins, would indicate the presence and / or degree of misfolding of the proteins. In some methods, the one or more misfolded proteins include transthyretin (TTR) and / or a TTR misfolding surrogate protein. In some of these methods, the TTR misfolding surrogate protein is TBG, APOB, or APOA1.

[0008] In some methods of the invention, the at least one peptide to be examined for amino acid solvent accessibility and solvent exclusion is produced by digestion of the biological sample with a protease. In some methods, solvent accessibility and solvent exclusion are determined by differential labeling of the one or more amino acid residues in solvent accessible and solvent excluded states. In some methods, the one or more amino acid residues in solvent accessible state is labeled with a first label, and the one or more amino acid residues in solvent excluded state is thereafter labeled with a second label after denaturing the at least one peptide already labeled with the first label. In some methods, the one or more amino acid residues to be quantified for solvent accessibility and solvent exclusion are lysine residues.

[0009] Some methods of the invention utilize a biological sample that is obtained from a subject having, suspected of having, or suspected of developing ATTR amyloidosis. In some embodiments, the biological sample obtained from the subject is a plasma sample, a serum sample or a whole blood sample. In some methods, the ATTR amyloidosis is wildtype (wt) TTR amyloidosis or V122I TTR amyloidosis. In some embodiments, the at least one peptide to be examined encompasses a destabilized or aggregated region of TTR that is associated with ATTR amyloidosis. In some methods, the at least one peptide to be examined encompasses TTR β-strand a, b, or c. In some methods, the TTR peptide to be examined encompasses one or more amino acid residues selected from Lys35, Lys55, Lys68and Lys146, wherein the amino acid numbering is based on UniProt ID No. P02766. In various embodiments, solvent accessibility and solvent exclusion for the one or more amino acid residues are quantified via mass spectrometry analysis.

[0010] In a related aspect, the invention provides methods for diagnosing TTR amyloidosis in a subject. These methods entail (a) obtaining a biological sample from a candidate subject, and (b) quantifying solvent accessibility and solvent exclusion for one or more amino acid residues in one or more misfolded proteins in the biological sample. In these methods, different molecules of each of the one or more misfolded proteins contain the one or more amino acid residues in both solvent accessible and solvent excluded states. By detecting a decrease of the ratios of solvent accessibility to solvent exclusion for the one or more amino acid residues in the one or more misfolded proteins, relative to the ratios for the same amino acid residues in the same proteins from amyloid-negative control subjects, TTR amyloidosis in the candidate subject is positively diagnosed. In some of these methods, quantification of solvent accessibility and solvent exclusion is performed after (a) digesting the biological sample with a protease to generate from each of the one or more misfolded proteins one or more peptides containing the one or more amino acid residues, and (b) differentially labeling the one or more amino acid residues in solvent accessible and solvent excluded states. In some methods, the one or more misfolded proteins include transthyretin (TTR) and / or a TTR misfolding surrogate protein. In some methods, the one or more amino acid residues include at least 2, 3, 4 or more different residues in the TTR protein. In some of these methods, the one or more amino acid residues are lysine residues. In some embodiments, the one or more amino acid residues to be quantified for solvent accessibility and solvent exclusion include Lys35, Lys55, Lys68and Lys146of the TTR protein, with the amino acid numbering being based on UniProt ID No. P02766.

[0011] In another aspect, the invention provides methods for monitoring treatment effect in a TTR amyloidosis subject undergoing treatment. These methods entail (a) obtaining a first blood sample from the subject prior to treatment, (b) detecting and quantifying one or more misfolded proteins in the first blood sample, (c) obtaining a second blood sample from the subject during or subsequent to treatment, (d) detecting and quantifying the same one or more misfolded proteins in the second blood sample, and (e) comparing the amount of the one or more misfolded proteins in the two blood samples. In these methods, misfolded proteins in the blood samples are detected and quantified by (i) generating from each of the blood samples at least one peptide containing one or more amino acid residues in both solvent accessible and solvent excluded states, (ii) quantifying solvent accessibility and solvent exclusion for the one or more amino acid residues for each of the blood samples, and (iii) calculating the ratio of solvent accessibility to solvent exclusion for the one or more amino acid residues. A significant increase in the ratio calculated for the second blood sample over the ratio calculated for the first blood sample indicates a positive treatment effect in the subject. In some of these methods, the one or more misfolded proteins include TTR protein and / or a TTR misfolding surrogate protein. In some methods, the one or more amino acid residues to be quantified for solvent accessibility and solvent exclusion include Lys35, Lys55, Lys68and Lys146of the TTR protein, with the amino acid numbering based on UniProt ID No. P02766.

[0012] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. DESCRIPTION OF THE DRAWINGS

[0013] Figure 1. Solvent accessibility for TTR lysine residues 55, 68 and 146 is given in percentage for samples from healthy control patients, cardiomyopathy patients with ATTR caused by aggregation of wt TTR (TTR_cardiomyopathy) and cardiomyopathy patients with ATTR caused by the V122I mutation in TTR. The difference in solvent accessibility clearly distinguishes patients from healthy controls, except at Lys 146, which does not show any significant difference and serves as internal control.

[0014] Figure 2. CPP workflow and concept. The e-amine groups of solvent-exposed lysine residues are labeled in their native state with a small isotope-coded, covalent label within 20 seconds in fresh or frozen plasma. After digestion with proteases, lysines that were solvent-excluded in the native state are labeled with the same covalent, but differently isotope-encoded label. The ratio of the two isotopes is measured in a mass spectrometer anddirectly reflects the ratio of solvent accessibility to solvent exclusion of a lysine, which is informative of the fold of a protein. A misfold is measured as a change in the isotope ratio compared to a control sample.

[0015] Figure 3. Misfolding of TTR and TBG was detected in plasma samples of ATTR patients, but not in samples from healthy controls, as indicated by significantly reduced solvent accessibility at two TTR sites (Lys 55 and Lys 68) and one TBG site (Lys64). TBG serves as an additional marker to distinguish ATTR patients from healthy control patients. Solvent accessibility at TTR Lys 146 and Lys 218 in TBG was not altered between healthy controls and ATTR patients and served as an internal control measurement. Statistical significance was analyzed by One-Way Anova with Benjamini-Hochberg-Krieger correction.

[0016] Figure 4. A difference in solvent accessibility of a lysine can also be expressed as a difference in the ratio of the light to the heavy isotope as exemplified in this blinded study with ATTR patients. The results indicated that misfolding of TTR distinguished healthy control from ATTR-CM patients and those on tafamidis treatment. Statistical significance was analyzed by One-Way Anova with Benjamini-Hochberg-Krieger correction.

[0017] Figure 5. Altered solvent accessibility of surrogate markers APOA1 and APOB in patients with ATTR caused either by aggregation of wt TTR (labeled TTR_cardiomyopathy) or by the V122I TTR mutation (V122I_TTR_cardiomyopthy) when compared to healthy control patients.

[0018] Figure 6. Schematic depicting presence and increase of misfolded TTR protein in asymptomatic ATTR patients as modeled from actual patient data. It is expected that misfolded TTR is already present in asymptomatic patients, that the quantity of misfolded TTR will rise over time even before symptoms arise, likely around 10-20 years before conventional diagnostics with a PyP scan can detect TTR aggregates.

[0019] Figure 7. Increase of misfolded TTR protein over time as read out as increasing amount of heavy TTR.

[0020] Figure 8. Examples of patient profiles with progressing ATTR as illustrated by increasing amounts of misfolded TTR (top panel) and patients with low levels of misfolded TTR that are not progressing yet (lower panel). DETAILED DESCRIPTION I. Overview

[0021] Currently no method is available to diagnose the most common form of ATTR amyloidosis, which is caused by wildtype TTR, outside of specialized amyloidosis centers. Inpart because this is still perceived to be a relatively rare disease, too many physicians have a low suspicion level for systemic amyloidosis. Even if the physician is aware that amyloidosis is a possible diagnosis, these patients’ initial symptoms can mimic a number of other more common diseases and there is no single diagnostic method that is non-invasive and easy to apply currently. Instead, currently diagnostic methods for amyloidosis are invasive and rely on the detection of amyloid fibrils (e.g., by Congo Red staining, immunohistochemistry, or via mass spectrometry). Amyloid fibril detection, still considered the diagnostic gold standard, is generally combined with organ damage detected by echocardiographic and / or neurophysiological studies to make a diagnosis. As a direct result, diagnosis is often made later in the course of disease. This is problematic, as currently available therapies for the TTR amyloidosis – liver transplant and kinetic stabilizer (tafamidis and diflunisal) administration – have proven to be more effective when used early in the course of FAP.

[0022] The present invention is predicated in part on the studies undertaken by the present inventors to develop assays for detecting misfolded TTR, which can enable noninvasive, point-of-care, and early diagnostic methods for identifying amyloidosis patients. As detailed herein, the inventors obtained plasma samples from subjects with wt TTR amyloidosis or V122I TTR amyloidosis. Solvent accessibility of several specific amino acid residues (e.g., surface Lys residues) in TTR peptides or peptides of surrogate protein markers (e.g., thyroxine-binding globulin) from the patient plasma samples and also samples from healthy control subjects were examined. The inventors then quantified solvent accessibility and solvent exclusion of the examined amino acid residues, followed by calculation of the ratios of solvent accessibility and solvent exclusion for each of the examined residues. It was found that there is a statistically significant decrease in solvent accessibility or in the calculated ratios for one or more of the residues in the TTR and / or surrogate proteins of patient plasma samples, relative to the ratios for the same residues in the same proteins of control samples.

[0023] In accordance with these discoveries, the present invention blood or tissue-based molecular diagnostic tests for identifying amyloidosis patients or for monitoring disease status in patients undergoing treatment for TTR amyloidosis. The diagnostic test and assays described herein for quantifying misfolded TTR or surrogate proteins in patient plasma are readily applicable to aiding physicians in point-of-care diagnosis and in following the response to particular therapies. They can be employed widely to screen cardiomyopathy patients for ATTR amyloidosis. In particular, methods of the invention can shorten the time for diagnosis of ATTR amyloidosis, possibly preventing worse outcomes and progression ofthe disease, or even onset of the disease. Such early diagnostic tests are particularly advantageous, as treatments for ATTR amyloidosis (such as tafamidis) are available that have been shown to significantly slow the disease and evidence exists that early treatment with the drug is more effective than late treatment.

[0024] Methods of the invention are readily suitable for identifying subjects with ATTR amyloidosis. The diagnostic methods can also be used to assess whether a given subject should undergo further testing or treatment for ATTR amyloidosis. Further encompassed by the invention are methods of using the diagnostic assays described herein to determine whether a treatment should be given patients diagnosed with ATTR, as well as applications of the assays for disease monitoring. Unless otherwise stated, the present invention can be performed using standard procedures, as described, for example in Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13: 978-0121821906); U.S. Pat. Nos. 4,965,343, and 5,849,954; Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol.57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998). The following sections provide additional guidance for practicing the compositions and methods of the present invention. II. Definitions

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (eds.), Oxford University Press (revised ed., 2000); Dictionary ofMicrobiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3PrdP ed., 2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4PthP ed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0026] The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[0027] As used herein, the term "amino acid" of a peptide refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Unless otherwise noted, the TTR derived probe peptides of the invention may encompass derivatives or analogs which have been modified with non-naturally coding amino acids.

[0028] Natural amino acids that make up a polypeptide or protein can be grouped according to what their side chains are like. Based on the propensity of the side chain to be in contact with polar solvent like water, it may be classified as hydrophobic, polar or charged. Hydrophobic amino acid refer to amino acids or residues that have hydrophobic side chains. These include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). These side chains are composed mostly of carbon and hydrogen, have very small dipole moments, and tend to be repelled from water. This fact has important implications for proteins' tertiary structure. Six amino acids have side chains that are polar but not charged. These are serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), glutamine (Gln), and tyrosine (Tyr). These amino acids are usually found at the surface of proteins. Charged amino acid residues include lysine (Lys), arginine (Arg), aspartate (Asp) and glutamate (Glu).

[0029] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0030] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0031] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0032] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0033] For polypeptide or peptide sequences, “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched side chains(e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0034] As used herein, a “derivative” of a reference molecule (e.g., a TTR derived probe peptide disclosed herein) is a molecule that is chemically modified relative to the reference molecule while substantially retaining the biological activity. The modification can be, e.g., oligomerization or polymerization, modifications of amino acid residues or peptide backbone, cross-linking, cyclization, conjugation, fusion to additional heterologous amino acid sequences, or other modifications that substantially alter the stability, solubility, or other properties of the peptide.

[0035] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0036] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482c, 1970; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.48:443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI); or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402, 1977; and Altschul et al., J. Mol. Biol.215:403-410, 1990, respectively.

[0037] Other than percentage of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptideencoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0038] As used herein, a diagnostic moiety or label moiety is a functional group, compound, molecule, substituent, or the like, that can enable detection of a target molecule (e.g., a protein or peptide) to which it is conjugated, either via covalent bonding or non- covalent attachment. It can provide a detectable biological or physiochemical signal that allows detection via any means, e.g., fluorescence, phosphorescence, absorbance, luminescence, chemiluminescence, radioactivity, colorimetry, magnetic resonance, or the like. The detectable signal provided by the diagnostic moiety or label moiety can be directly due to a biochemical or physiochemical property of the moiety (e.g., a fluorophore) or indirectly due to its interaction with another compound or agent. For labeling a peptide derived from TTR or a surrogate marker, the diagnostic moiety or label moiety does not encompass peptide sequences that are naturally present in the TTR or surrogate protein. Typically, the diagnostic moiety or label moiety used in the invention is a small functional group or small organic compound. In various embodiments, the employed diagnostic moiety or label moiety has a molecular weight of less than about 1,000 Da, 750 Da, 500 Da or even smaller.

[0039] Amyloidosis is a rare disease that results from the buildup of misfolded proteins into a spectrum of aggregates including oligomers and amyloid fibrils. When proteins that are normally dissolvable in water misassemble into amyloid fibrils, they become insoluble and deposit in organs or tissues, disrupting normal function. The type of protein that is misfolded and misassembled, and the organ or tissue in which the misfolded aggregated proteins are deposited determine the clinical manifestations of the specific amyloidosis. There are four main types of systemic amyloidosis, each due to the deposition of a specific protein. The most common type is AL amyloidosis, caused by the deposition of light chain proteins produced by plasma cells in different disease states. The second most common is AA amyloidosis due to the accumulation of S amyloid A protein or SAA, which occurs in association with chronic infections - e.g. tuberculosis - or inflammatory illnesses such asrheumatoid arthritis. The third and the fourth type are due to the deposition of a genetically defective or normal form of a protein called transthyretin respectively.

[0040] Transthyretin (TTR) is a 127-amino acid β-sheet-rich protein that folds and assembles into a tetramer in the endoplasmic reticulum of liver, choroid plexus, and retinal pigmented epithelial cells, from which TTR is secreted into the plasma, cerebrospinal fluid, and eye, respectively. Rate-limiting TTR tetramer dissociation, and relatively fast monomer misfolding leads to TTR aggregation associated with a number of systemic amyloid diseases, which are autosomal dominant diseases collectively referred to as the TTR amyloidosis (ATTR). These maladies are named after one of several aggregate structures, namely the cross-β sheet amyloid fibril, that forms in the extracellular space of tissue. There is evidence from other amyloid diseases that different amyloid structures can form from the same protein, and these are referred to as strains associated with distinct clinical phenotypes. Wild type TTR amyloidosis (ATTR-WT; previously known as senile systemic amyloidosis), affects approximately 10-15% of individuals older than 65 years of age. ATTR-WT manifests predominantly as a cardiac disease, but the involvement of other organ systems, including the nervous system, is increasingly recognized in this malady that is being diagnosed with increasing frequency.

[0041] Non-native or misfolded TTR oligomer refers to soluble oligomers or other aggregate types of TTR which are formed as a result of the misfolding and mis-assembly of TTR monomers. Unlike normal circulating TTR which are tetrameric, these TTR aggregates results from misfolding and mis-assembly of monomeric TTR molecules released by dissociation of the normal tetrameric TTR.

[0042] TTR amyloid diseases (TTR amyloidosis or ATTR amyloidosis) are diseases that are caused by amyloid deposits made up of transthyretin (TTR). There are a few distinct different types of ATTR amyloidosis, including (1) familial amyloid polyneuropathy (FAP) which is hereditary and can overlap with FAC, (2) familial amyloid cardiomyopathy (FAC) which is hereditary and can overlap with FAP), and (3) senile systemic amyloidosis or wild- type ATTR amyloidosis which is not hereditary and it mostly causes a cardiomyopathy. In the case of the familial TTR amyloidosis, caused by the dissociation of TTR tetramers comprising WT and / or mutant TTR subunits, followed by their misfolding and mis-assembly, the clinical heterogeneity is even more marked. The familial TTR amyloidosis can manifest with different primary phenotypes, including heart failure with a preserved ejection fraction, in a disease called familial amyloid cardiomyopathy (FAC) caused by inherited TTR mutations like Val122Ile (present in 4 % of individuals of African descent). Individualsinheriting other mutations, e.g., Val30Met, can present with a predominantly neuropathic disease, a long fiber neuropathy with autonomic nervous system involvement, in a disease called familial amyloid polyneuropathy (FAP).

[0043] As used herein, the term click chemistry refers to the copper(I)-catalyzed [3+2]- Huisgen 1,3-dipolar cyclo-addition of terminal alkynes and azides leading to 1,2,3-triazoles. It may also refer to a copper free variant of this reaction that might also be used. (J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang, I. A. Miller, A. Lo, J. A. Codelli, C. R. Bertozzi, Proc. Natl. Acad. Sci. U.S.A.2007, 104, 16793.).

[0044] Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures. A mass spectrum is a type of plot of the ion signal as a function of the mass-to-charge ratio. These spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical identity or structure of molecules and other chemical compounds. In a typical MS procedure, a sample, which may be solid, liquid, or gaseous, is ionized, for example by bombarding it with a beam of electrons. This may cause some of the sample's molecules to break up into positively charged fragments or simply become positively charged without fragmenting. These ions (fragments) are then separated according to their mass-to-charge ratio, for example by accelerating them and subjecting them to an electric or magnetic field: ions of the same mass-to-charge ratio will undergo the same amount of deflection.[1]The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier. Results are displayed as spectra of the signal intensity of detected ions as a function of the mass-to-charge ratio. The atoms or molecules in the sample can be identified by correlating known masses (e.g. an entire molecule) to the identified masses or through a characteristic fragmentation pattern.

[0045] Solvent accessibility (SA) or solvent accessible surface area (SASA) is a measure of the exposure of a residue to solvent and is a key feature of proteins for determining their folding and stability. See, e.g., Savojardo et al., Front. Mol. Biosci.2021; 7:7:626363. When the folding or stability of a protein changes, such as in a misfolding disease as ATTR where the protein forms aggregates, the SASA may also change for amino acids that are in regions undergoing the folding change, or that are involved in the aggregation. The same is true for any conformational change a protein may undergo. It is noted that not all amino acids in a misfolded region may change their SASA.

[0046] Tandem mass spectrometry (MS / MS) is a technique in instrumental analysis where two or more stages of analysis using one or more mass analyzer are performed with an additional reaction step in between these analyses to increase their abilities to analyze chemical samples.[1]A common use of tandem MS is the analysis of biomolecules, such as proteins and peptides. The molecules of a given sample are ionized and the first spectrometer (designated MS1) separates these ions by their mass-to-charge ratio (often given as m / z or m / Q). Ions of a particular m / z-ratio coming from MS1 are selected and then made to split into smaller fragment ions, e.g. by collision-induced dissociation, ion-molecule reaction, electron transfer dissociation, electron capture dissociation, surface induced dissociation, or photodissociation. These fragments are then introduced into the second mass spectrometer (MS2), which in turn separates the fragments by their m / z-ratio and detects them. The fragmentation step makes it possible to identify and separate ions that have very similar m / z-ratios in regular mass spectrometers. Selected product ions generated in MS2 can be further fragmented to produce another group of product ions (MS3) and so on (MSn).

[0047] As used herein, the term "peptide mimetic" or "peptidomimetic" refers to a derivative compound of a reference peptide (e.g., a TTR derived peptide disclosed herein) that biologically mimics the peptide’s functions. In some embodiments, a peptidomimetic derivative of a TTR derived probe peptide may have at least 25%, at least 50%, at least 75% or at least 90% of the misfolded TTR oligomer-binding activity of the reference peptide.

[0048] The term “subject” includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.

[0049] As used herein, the term "variant" refers to a molecule (e.g., a pre-defined TTR peptide described herein) that contains a sequence that is substantially identical to the sequence of a reference molecule. In some other embodiments, the variant differs from the reference molecule by having one or more conservative amino acid substitutions but substantially retains the biological activity of the reference molecule. III. Detecting misfolded TTR and surrogate proteins via quantifying SA / SE ratios

[0050] Transthyretin (TTR) is a 127-amino acid (~55 kDa) homotetrameric protein present in serum and cerebral spinal fluid. The function of TTR is to transport L-thyroxine (T4) and holo-retinol binding protein (RBP). TTR is one of greater than 20 nonhomologousamyloidogenic proteins that can be transformed into fibrils and other aggregates leading to disease pathology in humans (ATTR amyloidosis). These diseases do not appear to be caused by loss of function due to protein aggregation. Instead, aggregation appears to cause neuronal / cellular dysfunction by a mechanism that is not yet clear. Under denaturing conditions, rate limiting wild type TTR tetramer dissociation and rapid monomer misfolding enables mis-assembly into amyloid that causes wildtype TTR amyloidosis (WT-ATTR). Dissociation and misfolding of one of more than 120 TTR variants results in hereditary ATTR amyloidosis, including familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC).

[0051] The invention provides tissue sample based test, also termed Amyfold assay herein, for quantitatively detecting misfolded TTR and related surrogate proteins. In general, the diagnostic tests employ a biological sample, such as a blood or tissue sample, that is obtained from a subject in need of a diagnostic test for ATTR amyloidosis. The subject can be one who is suspected to have or develop ATTR amyloidosis. In some embodiments, the subject can be one who has an unspecified cardiovascular disease. The subject can also be one who has previously been diagnosed with ATTR amyloidosis. The subject can also be one who is undergoing a treatment for ATTR amyloidosis. In general, the diagnostic assays of the invention can utilize a biological sample obtained from any type of tissue. In various embodiments, the biological sample can be a plasma sample, a serum sample, a whole blood sample, which can be either fresh or frozen. In some other embodiments, a tissue sample that can be obtained from, e.g., heart biopsies, liver biopsies, or cerebrospinal fluid (CSF). Proteins in the sample are then digested with proteases to generate specific peptides. For misfolded proteins (TTR or a surrogate protein), the digestion will yield one or more peptides that encompass the misfolded region (destabilized or aggregated regions). If necessary, the digested peptides manifesting conformation change of the protein (e.g., TTR) can be purified thereafter, e.g., using 3D BioAnalytiX platform as exemplified herein. Typically, one or more amino acid residues of different molecules of each of such digested peptides from misfolded proteins are present in both solvent accessible and solvent excluded forms. Such amino acid residues in misfolded regions in many misfolded proteins are known and characterized in the art, esp. residues that can be readily detected via routinely practiced assays (e.g., Lys residues detected via MS as exemplified herein). Using TTR as an example, misfolded domains of TTR are known to span at least residues L32 to L75 (UniProt ID P02766). Depending on the specific method of digestion, a number of TTR peptides containing the relevant Lys residues can be generated for measuring solvent accessibility and solvent exclusion. As illustration,examples of peptides from digestion of TTR with chymotrypsin that contain Lys35, Lys55, Lys68or Lys146are shown in SEQ ID NOs:1-10. Understandably, different peptides can be generated that contain one or more such residues, if a different digestion method is used (e.g., digestion via other proteases such as elastase, trypsin, proteinase K, AspN or GluC).

[0052] In addition or as alternative to measuring residues in TTR peptides, peptides digested from surrogate proteins that is indicative of TTR misfolding can be analyzed. For example, methods of the invention can quantify solvent accessibility and solvent exclusion of peptides similarly digested from a surrogate biomarker such as thyroxine-binding globulin (TBG, aka SERPINA7; UniProt ID P05543) as exemplified herein (e.g., Figure 3). As described below, the misfolded region in this surrogate protein, which is known to bind to TTR, was identified to encompass Lys64and span residues A36 to F82. Examples of digested peptides from this protein that can be examined for solvent accessibility and solvent exclusion include TVETPDKNIF (Lys64; SEQ ID NO:11) and TVETPDKNIFF (Lys64; SEQ ID NO:12). Beyond thyroxine-binding globulin, any other proteins present in the measured biological sample (e.g., a plasma sample) that also misfold in correlation with TTR misfolding and is indicative of TTR misfolding, which are collectively termed herein “TTR misfolding surrogate proteins”, can also be included as surrogate markers in the diagnostic methods of the invention. Examples of such additional surrogate proteins or markers include TBG (SERPINA7), APOB (UniProt ID P04114), and APOA1 (UniProt ID P02647) as exemplified herein. Digested peptides from these surrogate proteins (e.g., APOB and APOA1) that can be examined for solvent accessibility and solvent exclusion are also exemplified herein. Digested TTR peptides containing Lys in solvent accessible and solvent excluded states Lys 35 MVKVLDAVRGSPAINVAVH (SEQ ID NO:1) Lys 35 MVKVLDAVRGSPAINVAVHVF (SEQ ID NO:2) Lys 55 RKAADDTWEPF (SEQ ID NO:3) Lys 55 VFRKAADDTWEPF (SEQ ID NO:4) Lys 68 ASGKTSESGEL (SEQ ID NO:5) Lys 68 ASGKTSESGELHGL (SEQ ID NO:6) Lys 68 ASGKTSESGELHGLTTEEEF (SEQ ID NO:7) Lys 68 ASGKTSESGELHGLTTEEEFVEGIY (SEQ ID NO:8) Lys 146 SYSTTAVVTNPKE (SEQ ID NO:9) Lys 146 STTAVVTNPKE (SEQ ID NO:10)Digested APOB peptides containing Lys in solvent accessible and solvent excluded states Lys 1702 SLDGKAAL (SEQ ID NO:11) Lys 2100 KHINIDQF (SEQ ID NO:12) Digested APOA1 peptides containingsolvent excluded states Lys 206 EALKENGGARL (SEQ ID NO:13)

[0053] Methods of the invention entail quantitative examination of misfolding of the digested proteins (e.g., TTR) by measuring solvent accessibility and solvent exclusion for one or more amino acid residues in one or more digested peptides. Ratios of solvent accessibility to solvent exclusion (“SA / SE” ratios) for each of the measured amino acid residues can then be compared to negative control ratios for the same residue from the same but normally folded protein (e.g., TTR from healthy subjects or recombinantly produced normal TTR), and / or positive control ratios for the same residue from the same but known misfolded protein (e.g., misfolded TTR generated recombinantly or isolated from ATTR amyloidosis patients). The degree of departure of the calculated ratios from the control ratios will indicate the severity of the misfolding of the examined protein in the biological sample (e.g., TTR). It is to be noted that, while the invention is primarily described by calculating a reduction or decrease of SA / SE ratios for one or more amino acid residues as an indicator of a misfolded protein, a measured reduction of SA / SE ratio encompasses an increase of solvent exclusion to solvent accessibility ratio (“SE / SA ratio”). In other words, a decrease of SA / SE ratio and an increase of SE / SA ratio are used interchangeably in the practice of the methods of the invention.

[0054] In various embodiments, the diagnostic tests of the invention involve examining digested peptides from one or more proteins in the biological sample. In some preferred embodiments, the examined one or more proteins include TTR. For each examined protein, one or more digested peptides, each containing one or more amino acid residues that are present in both solvent accessible and solvent excluded states, are subject to assays (e.g., MS assays exemplified herein) to quantify SA / SE ratio for each of the amino acid residues. In some embodiments, a positive diagnostic test is indicated by a significant decrease of the SA / SE ratio for one examined amino acid residue. In some embodiments, a positive diagnostic test is indicated by a significant decrease of the SA / SE ratios for 2, 3, 4 or more examined amino acid residue. In the latter embodiments, the one or more examined aminoacid residues can be present in one digested peptide or multiple digested peptides from the same protein (e.g., TTR). Alternatively, they can be present in digested peptides from multiple misfolded proteins in the biological sample.

[0055] Detection of misfolded TTR proteins or surrogate proteins via methods of the invention is based on a measured solvent accessibility to solvent exclusion ratio for the candidate subject relative to a background or control ratio measured via the same test. Using TTR as an example, the background or control ratio is typically determined with the same type of biological sample that is known not to contain misfolded TTR oligomer (e.g., biological sample from healthy subjects. Alternatively, the control ratio can be based on recombinantly expressed TTRs, as described herein. If the calculated ratio does not differ significantly from the control ratio, the outcome of the diagnostic assay is considered negative. On the other hand, if there is a significant departure between the measured ratio for a biological sample from a candidate subject and the control ratio, it indicates a positive outcome of the diagnostic test. Additionally or alternatively, the assays can further include comparing the measured ratio for the candidate subject with a positive standard or control ratio. The latter is determined with biological samples from a group of subjects known to be affected by a specific type of TTR amyloidosis. In some of these embodiments, the controls are age-matched subject, e.g., within 0-10 years of age as the candidate subject to be tested. A positive diagnosis is obtained if there is no significant difference between the measured ratio and the positive control ratio. For example, a positive diagnosis can be established by a measured ratio that is comparable to or falls within the range of positive control ratio determined from a population of subjects affected by a TTR amyloidosis or from recombinantly produced misfolded TTR.

[0056] A departure is considered significant or substantial if the measured ratio falls outside the range typically observed in unaffected subjects due to inherent variation between subjects and experimental error. For example, in some methods, a departure can be considered significant if a measured ratio does not fall within the mean plus one standard deviation of ratios in a control population. Depending on the specific misfolded protein and / or the examined amino acid residue in the specific digested peptide, the exact degree of departure to be considered significant can vary. In general, protein databases (e.g., MS databases) contain sequence and other structural information on a great number of misfolded proteins, including misfolded regions and key amino acid residues therein. Such information allows one to readily determine whether a measured solvent accessibility to solvent exclusion ratio for an amino acid residue in accordance with the present invention significantly orsubstantially departs from the normal range observed with the same residue in the same but properly folded protein. Thus, using examined residues in TTR as examples, a significant departure occurs if the measured ratio is at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more lower than the control ratio. For other misfolded proteins, a significant departure from control or normal range may require a bigger reduction of the calculated ratio (e.g., for surrogate protein thyroxine-binding globulin exemplified herein). It is further noted that the extent of departure between a measured ratio and a background or control ratio in a control population also provides an indicator of the probable accuracy of the diagnosis, and / or of the severity of the disease being suffered by the subject. IV. Measuring solvent accessibility and solvent exclusion of amino acids

[0057] As described herein, the degree of solvent accessibility of an amino acid residue in a digested peptide refers to the percentage of the molecules of the peptide containing the residue in solvent exposed state, and the degree of solvent exclusion of an amino acid residue in a digested peptide refers to the percentage of the molecules of the same peptide containing the residue in solvent excluded state. Alternatively, the degree of solvent accessibility of an amino acid residue in a digested peptide can also refer to the percentage of normalized area of the residue in solvent accessible state, and the degree of solvent exclusion of an amino acid residue in a digested peptide can also refer to the percentage of normalized area of the residue in solvent excluded state. As described herein, the normalized surface area for both states of a given amino acid residue can be readily measured via, e.g., MS analysis. Depending on the misfolded proteins and the measuring assays, solvent accessibility and solvent exclusion of different types of amino acid residues present in the digested peptides can be analyzed. In some preferred embodiments, the analyzed one or more amino acid residues in the digested peptides from the misfolded proteins are lysine residues.

[0058] Using misfolded TTR as exemplification, Lys residues in the digested peptides that are present in both solvent accessible state and solvent exclusion state can be readily measured with MS or other assays to determine their degrees of solvent accessibility and solvent exclusion and / or to calculate the ratio of solvent accessible to solvent exclusion. In various embodiments, the TTR peptides to be analyzed include peptides containing residue Lys55and / or Lys68, and peptides encompassing the destabilized or aggregated regions of TTR such as peptides containing residue Lys35or beta strands a, b, c, as well as peptides outside these regions that display a changed solvent accessibility of amino acids. Upon purification, the TTR derived peptides can be subject to differential labeling of the appropriate amino acidresidues in both solvent accessible and solvent excluded states. In various embodiments, the amino acid residues in the TTR peptide for differential labeling and subsequent measurement of solvent accessibility and solvent exclusion include lysine 35, lysine 55 and lysine 68. The amino acid numbering is based on TTR protein sequence with UniProt Accession Number P02766 (Figure 1). Due to substantial structural identity and sequence homology, consensus residues from other TTR variants or orthologs can be readily identified via alignment.

[0059] Any methods known in the art that are suitable for labeling a specific Lys residue or any other amino acid present in the misfolded regions of the misfolded protein (TTR or a surrogate protein) in plasma proteins or other tissue samples can be employed and adapted for the differential labeling of the digested peptides, e.g., at residues Lys35, Lys55 or Lys68 exemplified herein. The labeling is intended to generate a detectable signal from the peptides. In various embodiments, the detectable signal can be based on radioactivity, fluorescence, electrical conductivity, size, and the like. The signal may be directly or indirectly detectable. After the differential labeling, the amount and / or degree of solvent accessible residues (which correspond to “peptides in solvent accessible state”) and solvent excluded residues (which correspond to “peptides in solvent excluded state”) can then be quantified. The quantification can be performed with methods routinely practiced in the art. In some embodiments, a mass spectrometric method is employed to quantify peptides in solvent accessible state and peptides in solvent excluded state, as exemplified herein. In some embodiments, the ratio of solvent accessibility to solvent exclusion can be determined via a parallel reaction monitoring (PRM) assay. In such a PRM assay, the ratio of solvent accessibility to solvent exclusion is measured precisely by selectively measuring pre-defined TTR peptides only, as opposed to peptides from all plasma proteins present in the obtained biological samples. After quantification of the peptides in the two states, the ratios of peptides with solvent accessibility to peptides with solvent exclusion for one or more of the labeled amino acid residues (e.g., Lys55 or Lys68), which indicate the degree of misfolded TTR proteins, can then be calculated.

[0060] As noted above, the solvent accessibility / solvent exclusion ratio can be based on the number of peptide molecules containing the labeled residue in solvent accessible state vs. molecules containing the labeled residue in solvent excluded state. In some other embodiments, the ratio can be based on the normalized area ratio themselves as measured from analyses (e.g., MS spectrometry) of the two states of the differentially labeled sample, as detailed below. Peptides in solvent exclusion state and peptides in solvent accessibility state can be determined in one analysis (e.g., a mass spectrometry scan). In theseembodiments, a parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) can be employed to ensure that peptides encompassing the different Lys residues are all selected by the MS device for identification and quantification. Again using TTR as exemplification, TTR peptides in the two states can be quantified by determining the area under the peak for the light and the heavy isotope labeled precursor ion in an MS1 scan. In some other embodiments, TTR peptides in the two states can be quantified by measuring the intensity of the light and heavy isotope label containing fragment ions in an MS2 or MS3 (MSn) scan, either using the intensities directly to arrive at the ratio or their area under the peak. In some other embodiments, they can also be quantified via PRM, MRM or selected reaction monitoring (SRM). MRM and SRM are methods used in tandem mass spectrometry in which an ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ions is selected in the second mass spectrometer stage for detection. In still some embodiments of the invention, other mass spectrometry based methods such as fast photochemical oxidation of proteins (FPOP), limited proteolysis (LiP), or stability of proteins from rates of oxidation (SPROX) can be used to detect a change in TTR conformation. See, e.g., Johnson et al., J. Biol. Chem.2019; 294:11969-11979; Li et al., Acc. Chem. Res.2018; 51:736-744; Holfeld et al., Methods Mol. Biol.2023; 2554:69-89; Malinovska et al., Nature Protocols 2023; 18:659- 682; Strickland et al., Nat. Protoc.2013; 8:148-161; and West et al., Anal. Chem.2008; 80:4175-85..

[0061] As a further exemplification, Figure 2 illustrates experimental and mass spectrometric workflow depicting the two labeling reactions and quantification of solvent accessibility or solvent exclusion by using either MS1 or MS2 based methods. These include, e.g., data-dependent acquisition, data-independent acquisition or parallel reaction monitoring type acquisition for determining the ratio as measured by the ratio of the two isotopes. Specifically, solvent accessible amino acids in the TTR proteins or peptides from a blood sample are labeled with a dimethyl group by using formaldehyde and sodium cyanoborohydride to form a Schiff base. This is followed by attaching the methyl labels to the epsilon amino group of solvent accessible lysine residues, whereby both formaldehyde and sodium cyanoborohydride may or may not be isotope encoded using various deuterated or / and C13 labeled reagents. After quenching the labeling reaction, the most abundant plasma proteins are de-enriched as described in prior art, e.g., by precipitation with perchloric acid. The proteins are then denatured, reduced, and alkylated. Alternatively, the plasma sample can be prepared without de-enrichment of the most abundant plasma proteins, before the plasmaproteins are denatured, alkylated and reduced directly. Samples can then be loaded onto a filter device (e.g., the 3D BioAnalytiX device), washed, and digested with a protease. Alternatively, proteins in a sample may be further purified by state-of-the-art methods before digestion with a protease or directly digested with a protease. Upon digestion, amino acids in the resulting peptides not labeled in the first labeling reaction due to solvent exclusion, are labeled with a second label which differs in its isotope composition from the first label. The peptides resulting from the digestion are then analyzed by mass spectrometry. The analysis involves, e.g., separation of peptides on a chromatographic scale, and measurement and quantification of the peptides, which can include the use of isotope labeled standard peptides.

[0062] In some embodiments for monitoring disease progression or treatment effect, a solvent accessibility to solvent exclusion ratio is measured with the same type of biological sample (e.g., blood plasma) that is obtained from the candidate subject at multiple time points. In some embodiments for monitoring disease status or progression in a candidate subject, the time points can be every month, every other month, every 6 months, every year, or every other year. The monitoring period can last for a few years or for the remaining life of the candidate subject. In some of these embodiments, a decrease of over 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% or more of a measured ratio for residues in digested TTR peptides over time in the same subject is indicative of an increased likelihood of developing a TTR amyloid disease or an increased severity of disease. In some other embodiments for monitoring treatment effect, the time points can be, e.g., prior to treatment, during and / or after treatment. An increase of a measured ratio (e.g., by at least 1%, 2%, 3%, 4%, 5%, 6%, 7% or more) at a later time point relative to a measured ratio determined at a previous time point with the same type of sample from the same subject would suggest an improvement of disease symptoms or a positive treatment result. V. Assays for diagnosing and assessing disease status of ATTR amyloidosis

[0063] By quantifying digested peptides from TTR and / or other surrogate markers in both solvent accessible state and solvent excluded state (as indicated by the relevant examined amino acid residues), the diagnostic tests described herein can have many applications in disease diagnosis and disease monitoring related to ATTR amyloidosis. In some embodiments, the invention provides methods for diagnosing ATTR amyloidosis in a subject or a predisposition thereto. The methods entail obtaining a biological sample from a subject in need of testing, processing and labeling the sample, and then perform a diagnostic test with the sample as explained above to quantify digested peptides (e.g., TTR peptides) insolvent accessible state and peptides in solvent exclusion state. After the quantification of one or more relevant residues (e.g., the Lys residues in TTR as exemplified herein), a ratio of peptides in solvent accessible state vs. TTR peptides in solvent exclusion state is calculated, which provides an indicator as to whether the subject is suffering from or likely to develop ATTR amyloidosis. In some embodiments, the diagnostic tests can be performed with blood samples obtained from at risk population or asymptomatic patients. As detailed herein (e.g., Example 3), misfolded TTR protein thus detected and / or quantified in the blood samples can be used as an early diagnostic marker of ATTR amyloidosis or transthyretin amyloid cardiomyopathy (ATTR-CM).

[0064] As explained above, peptides containing each of the labeled residues in solvent accessible state vs. solvent excluded state can be readily measured with methods routinely practiced in the art. In some embodiments, a mass spectrometric method is employed to quantify labeled residues (e.g., certain Lys residues) in solvent accessible state and solvent excluded state. In some embodiments, residues in the two states can be determined via a parallel reaction monitoring (PRM) assay. In general, if the ratios of peptides in solvent accessibility to peptides in solvent exclusion for one or more of the measured residues fall out of the normal range of a control ratio, e.g., significantly lower than the control ratio, a positive diagnosis is made. The control ratio is typically established with the same residues in control samples that are obtained from healthy subjects not diagnosed with ATTR amyloidosis. To be considered a positive test, the extent of departure from the control depends on the specific examined protein and the method used for the measurement. Using TTR protein and the MS measuring method exemplified herein, a positive test typically requires a calculated SA / SE ratio for one or more examined amino acid residues that is less than 97%, 96%, or 95% of the control ratio. In some of these embodiments, a ratio of less than 94%, less than 93%, less than 92%, less than 91%, or less than 90% of a control ratio signifies a positive diagnosis. Once a subject is identified as a potential amyloidosis patient, additional diagnostic testing and / or therapeutic intervention may be prescribed. In some other embodiments, a reduction of the solvent accessibility to solvent exclusion ratio is calculated from the relative ratios based on the normalized surface areas that can be obtained from MS1 or MS2 measurements, or from the transitional ions that can be obtained from PRM, MRM or SRM assays.

[0065] In addition to disease diagnosis, the diagnostic methods described herein can also be employed in other settings. In some embodiments, they can be used in screening subjects for suitable participants in clinical trials. In some embodiments, the diagnostic methods canbe employed in assessing disease status or progression, and monitoring effect of treatments that TTR amyloidosis patients are undergoing. In some of these embodiments, the invention provides blood or tissue-based tests for monitoring treatment outcome in subjects who have been diagnosed with ATTR and are receiving therapeutic intervention. In these embodiments, the subject is subject to the diagnostic test at multiple time points (e.g., with the interval being a month, 2 months, 3 months, 6 months, a year, or longer) over a treatment period (e.g., 1 year, 2 years, 3 years, 4 years, 5 years, 7 years, 10 years or longer). An increase in the measured ratios over the period of time would indicate a positive outcome of the treatment. In some other embodiments, the methods can be employed to monitor disease progression in subjects already diagnosed with or at risk of developing ATTR amyloidosis. In these embodiments, the subject is subject to the diagnostic test at multiple time points (e.g., with the interval being a month, 2 months, 3 months, 6 months, a year, or longer) over a period of time (e.g., 1 year, 2 years, 3 years, 4 years, 5 years, 7 years, 10 years or longer). A decrease in the measured ratios over the period of time would indicate disease progression or worsening in the subject.

[0066] Subjects suitable for these methods of the invention can be TTR amyloidosis patients that are receiving any therapeutic treatment or intervention. Treatment of all types of amyloidosis is currently based on the following principles: reducing the supply of amyloid forming precursor proteins, and supporting the function of organs containing amyloid. All the TTR in the blood, which forms the amyloid deposits everywhere except in the eye and the blood vessels around the brain, is made in the liver. Thus, liver transplantation may be helpful for some patients with hereditary, variant ATTR amyloidosis, mainly for patients with FAP associated with the V30M mutation. In addition to liver transplantation, heart transplantation may also be an option, esp. for younger, otherwise healthy patients. Another proven treatment is kinetic stabilizers, e.g., tafamidis, that bind to the TTR tetramer slowing its dissociation, which is the rate-limiting step associated with TTR aggregation. Besides these options, treatment may also involve supporting the function of organs containing amyloid. Thus, in ATTR amyloidosis this may include treatment for heart disease, treatment of peripheral neuropathy symptoms, and treatment of autonomic neuropathy symptoms. Many medications can be used in these treatments. For example, medications that may help alleviate neuropathic pain include gabapentin, pregabalin and duloxetine. Other therapeutic regimens that may be available for treating TTR amyloid diseases include, e.g., drugs such as diflunisal and tafamidis, genetic based therapies, and antibody based therapies. The diagnostic tests of theinvention can be employed to assess disease status or monitor treatment effect in patients undergoing any of these treatments.

[0067] Diseases or disorders suitable for methods of the invention include ATTR amyloidosis and any other TTR amyloid diseases. In healthy people, normal “wild-type” TTR functions as a transporter of thyroid hormones and vitamin A (retinol) within the bloodstream. People with mutations in the TTR gene produce abnormal, amyloidogenic, “variant” TTR throughout their lives. Amyloid deposits consisting of abnormal “variant” TTR may cause familial amyloid polyneuropathy (FAP). This disease affects the peripheral nervous system, often the heart, and sometimes the kidneys and eyes. More than 120 variants of TTR have been observed to be associated with the amyloidosis, with the most common mutation worldwide being TTR Val30Met (V30M TTR) and TTR Val122Ile (V122I TTR). The most common type of FAP is associated with the Val30Met mutation in the TTR protein. It is thought to affect about 10,000 people in the world. Patients with this mutation often start to experience symptoms in their 30s. Sensory and autonomic neuropathies are the main symptoms; heart, kidneys and eye involvement are less common in this form of the disease.

[0068] Aggregates of abnormal “variant” TTR can also cause familial amyloid cardiomyopathy (FAC) (heart disease). In FAC patients, heart disease onset can be preceded by carpal tunnel syndrome. There is less peripheral nervous system disease, and other organs are usually not affected. FAC is seen most often in people of Afro-Caribbean or African American heritage, amongst whom a particular mutation known as Val122Ile (V122I) in the TTR protein is found to be very widespread. This mutation has been found in almost 4 in every 100 (4%) African Americans and almost 1 in 4 (23%) African Americans with a diagnosis of cardiac amyloidosis.

[0069] In addition to the FAP and FAC caused by aggregates of mutant TTR, “wild type” TTR may also be amyloidogenic, causing wild type ATTR amyloidosis (aka “senile systemic amyloidosis” or “SSA”). This is a slowly progressive disease. As detailed herein, wildtype ATTR amyloidosis is associated with many cardiovascular disorders. It is estimated that at least 10-15% of all heart failure is caused by wild type ATTR. The symptoms usually start after age 65. This condition is not hereditary, and it is far more common in men than in women. People with this condition do not have a mutation in the TTR gene and the amyloid fibrils are made up of normal, “wild type” TTR. These types of amyloid deposits are found at autopsy in 1 in 4 people over age 80, but in most cases they do not appear to cause any symptoms. Almost 50% of patients with wild type ATTR amyloidosis experience carpaltunnel syndrome – tingling and pain in the wrists, pins and needles in the hands. Carpal tunnel syndrome often appears 3-5 years before the symptoms of heart disease.

[0070] The diagnostic methods of the invention can be used in patients with any of these TTR amyloid diseases. While TTR amyloid diseases may be diagnosed by tissue biopsy, genetic testing and imaging studies, these currently available tests are often invasive and / or ineffective in early diagnosis of patients with very few symptoms, pre-symptomatic (immediately before developing any symptoms) patients or patients with wildtype ATTR amyloidosis. In contrast, as demonstrated herein, the diagnostic methods of the invention are non-invasive or minimally invasive. After obtaining a suspected biological sample (e.g., a blood sample) from a candidate subject, digesting the TTR proteins, and differentially labeling solvent accessible residues and solvent excluded residues in TTR peptides, and quantifying solvent accessible residues and solvent excluded residues, the ratio of solvent accessibility to solvent exclusion can then be readily calculated. All these steps can be performed via routinely practiced methods, e.g., as exemplified herein. If the ratio is significantly lower than that obtained from control samples from healthy subjects, the candidate subject is identified as one who likely has or is likely to develop a TTR amyloidosis disease or condition.

[0071] In various embodiments, TTR amyloidosis diseases or conditions suitable for diagnosing and / or monitoring with methods of the invention include, e.g., transthyretin- related hereditary amyloidosis (ATTR), hereditary conditions with predominant degeneration of the heart (cardiomyopathy), transthyretin amyloid cardiomyopathy (ATTR-CM), the peripheral nervous system (polyneuropathy), the central nervous system (meningocerebrovascular amyloidosis), and the eye (ocular amyloidosis). Also suitable for the invention are autosomal dominant neurodegenerative diseases such as familial amyloid polyneuropathy (FAP) and familiar amyloid cardiomyopathy (FAC). Unless otherwise noted, TTR amyloid diseases suitable for the diagnostic methods of the invention further include wild type TTR amyloidosis (ATTR-WT), aka senile systemic amyloidosis (SSA), which is not inherited and has aggregation formed from wild type TTR protein.

[0072] In some embodiments, the diagnostic methods of the invention can be employed in conjunction with other diagnostic methods known in the art. The diagnostic test of the invention can be administered to the candidate patients before or after the patients are examined with the known diagnostic methods. In some embodiments, suspected patients can be first screened with the non-invasive diagnostic methods of the invention. Upon a positive test with the diagnostic method of the invention, the patients with positive test results canthen be further subject to, e.g., genetic testing to confirm the existence of a known disease causing TTR mutant (e.g., V30M or V122I). The patients can also be additionally examined via an imaging test, e.g., echocardiogram, DPD scanning or cardiac MR scanning. EXAMPLES

[0073] The following examples are provided to further illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. Example 1: Detecting TTR misfolding by quantifying solvent accessibility and exclusion

[0074] The present invention relates to blood-based methods for detecting ATTR amyloidosis and related systems form performing the methods. The diagnostic methods described herein are based on the finding that solvent accessibility of certain TTR amino acids in blood changes in subjects with ATTR amyloidosis, in comparison to that in amyloid- negative control subjects. This is likely due to destabilization and aggregation of the TTR molecule in ATTR amyloidosis. Specifically, it was observed that, while on average the ratio of the solvent accessibility and solvent exclusion of amino acid residue Lys55 decreased by 8.37 +- 1.9 % in subjects with wt TTR amyloidosis and by 17 +- 2.2 % for subjects with V122I TTR amyloidosis, the ratio of solvent accessibility and solvent exclusion of amino acid residue Lys68 decreased by 8.3 +- 0.9% for subjects with wt TTR amyloidosis and by 12% +- 1.4% for V122I TTR amyloidosis (Figures 1 and 3). Importantly, the methods and systems described herein measure the solvent accessibility and solvent exclusion of amino acid residues in a TTR peptide with a high degree of precision, allowing for quantification of small differences in the ratio of the solvent accessibility and solvent exclusion of amino acid residues to be accurately quantified and therefore have clinical utility. As noted above, methods of the invention can be interchangeably described by measuring either a decrease of solvent accessibility to solvent exclusion (SA / SE) ratio or a corresponding increase of solvent exclusion to solvent accessibility (SE / SA) ratio. Other than percentage of solvent accessibility, the change of solvent accessibility of the relevant amino acid residues can also be described with the relative ratios measured from the surface areas. For example, a ratio of 1:2 is obtained when the integrated area under the peak of the MS1 or MS2 signal is twice as large for one label as for the other label. For one of the misfolded TTR peptides containing Lys 68 (SEQ ID NO:5), for example a log2 transformed ratio of less than 1.8 (solvent accessibility / solvent exclusion) was observed for most ATTR patients (median 1.4) anddifferentiated them from control patients (median 2.3) as well as patients on tafamidis treatment, which stabilizes TTR (Figure 4).

[0075] As exemplification, solvent accessibility and solvent exclusion of the relevant amino acid residues in TTR were determined by differential labeling of lysine residues by Covalent Protein Painting (Figure 2) and measuring the respective isotope signal in a mass spectrometer. See also, Bamberger et al., Structural Changes of the Proteome in Alzheimer's Disease. J Proteome Res 20, 2762-2771 (2021); and Son et al., Quantitative structural proteomics in living cells by covalent protein painting. Methods Enzymol 679, 33-63 (2023). Prior to analyzing, solvent accessible amino acids in proteins or peptides from a blood sample (whole blood, plasma or serum sample, either fresh or frozen) are labeled with a first label (in this case a dimethyl group by using formaldehyde and sodium cyanoborohydride to form a Schiff base and attach the methyl labels to the epsilon amino group of solvent accessible lysine residues, whereby both formaldehyde and sodium cyanoborohydride may or may not be isotope encoded using various deuterated or / and C13 labeled reagents). After quenching the labeling reaction, the most abundant plasma proteins are de-enriched, such as described in prior art, for example by precipitation by perchloric acid and proteins denatured, reduced, and alkylated. See, e.g., Viode et al., A simple, time- and cost-effective, high-throughput depletion strategy for deep plasma proteomics. Sci Adv 9, eadf9717 (2023). Alternatively, the plasma sample is prepared without de-enrichment of the most abundant plasma proteins and plasma proteins denatured, alkylated and reduced directly. Samples are then loaded onto a filter device (provided by 3D BioAnalytiX, Inc.), washed, and digested with a protease. Alternatively, proteins in a sample may be further purified by state-of-the-art methods before digestion with a protease or directly digested with a protease. Upon digestion, amino acids in the resulting peptides not labeled in the first labeling reaction due to solvent exclusion, are labeled with a second label which differs in its isotope composition from the first label. Peptides are then analyzed by mass spectrometry including separation of peptides on a chromatographic scale, and peptides are measured and quantified, which may include the use of isotope labeled standard peptides. Example 2: Detecting TTR misfolding by quantifying surrogate proteins TBG

[0076] In this study, we extended the solvent accessibility and exclusion analysis to thyroxine-binding globulin (TBG) as surrogate marker for transthyretin (TTR) misfolding. Solvent accessibility ratios of specific lysine residues in TBG were measured in plasma samples from subjects diagnosed with wildtype TTR (wt TTR) amyloidosis, alongsidecontrol subjects. Plasma samples were prepared as described above, and TBG measured simultaneously with TTR. The results indicated a significant decrease in the solvent accessibility of Lys64 in TBG. This is evidenced by a mean log2 ratio difference 1.01, with a mean log2 ratio of 2.33 for control samples and a mean log 2 ratio of 1.326 for ATTR-CM patients. These findings support the use of TBG as additional marker for assessing TTR misfolding. Example 3: Detecting TTR misfolding by examining additional surrogate proteins

[0077] To broaden the analysis of TTR misfolding, we investigated solvent accessibility in other surrogate markers, including APOB and APOA1. Plasma samples from amyloidosis patients revealed notable changes in the solvent accessibility ratios of key lysine residues in these proteins (Figure 5). APOB and APOA1 are frequently associated with amyloidogenic processes in neurodegenerative diseases. APOB demonstrated increased accessibility at Lys1702 (on average a 7.4 % increase for wt TTR and 13.3 % for V122I TTR) and decreased solvent accessibility at Lys 2100 (on average a 3.6% decrease for wt TTR and 5.2% for V122I TTR). APOA1 showed slightly increased solvent accessibility at Lys 206 (1.8 % for wt TTR and 3.2 % for V122I TTR). These reductions align with TTR misfolding events and provide additional tools for amyloidosis diagnosis, complementing direct TTR assessments.

[0078] In this study, the methodologies employed for quantification were identical with those used in Example 1, and measurements were made simultaneously for TTR, APOB, APOA1 and TBG (SERPINA7) in the same sample, ensuring reliable results across multiple surrogate markers.

[0079] Because the Amyfold assay directly detects the misfolding of TTR (the first pathogenic step in amyloid formation), it has the potential for early, even presymptomatic, diagnosis of ATTR-CM. At this stage, severe organ damage could still be prevented, and treatment is more effective. See, e.g., Elliott et al., Eur. J. Heart Fail.2023; 25:2060-4 ; and Garcia-Pavia et al., J. Card. Fail.2025; 31:525-5334. To demonstrate the potential of the Amyfold assay for this application, we used blinded plasma samples from patients with familial amyloidosis (hATTR) who are participating in a longitudinal study at the Cleveland Clinic, with a median age of 45. These patients carry various misfolding mutations in the TTR gene such as V122I, T60A, S77Y or V30M associated with ATTR-CM, and were presymptomatic, but are within 10 years of expected disease onset at study start in 2018.Most patients were seen for yearly visits after the study start and plasma samples were collected at each of those visits. The plasma samples are provided to us blinded and anonymized. We expect to see an increase in the levels of misfolded and aggregated TTR over time (Figure 6). Results from this study will inform follow up studies to also enable early diagnosis for spontaneous wt ATTR and incorporation of the test as an initial screening tool into a cardiac marker panel.

[0080] Preliminary results provide proof of concept that the Amyfold assay was able to detect misfolded TTR in still asymptomatic ATTR patients with different hereditary mutations that make TTR protein more prone to misfolding and was able to distinguish increase of misfolded TTR oligomers carrying either the mutation as well as increase of misfolded wt TTR in the same patient (Figure 7).

[0081] Furthermore, the assay can be used to distinguish progressing from non- progressing patients, so clinicians can decide who to treat and when to start treating patients. This unique capability of the test makes it suitable for screening at risk populations such as patients with suspected heart failure as well as for preventative screening (Figure 8). ***

[0082] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0083] All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method for detecting the presence and quantifying degree of misfolding of proteins in a biological sample, comprising (a) generating at least one peptide from one or more misfolded proteins present in the biological sample, with different molecules of the at least one peptide containing one or more amino acid residues in both solvent accessible and solvent excluded states, (b) quantifying solvent accessibility and solvent exclusion for each of the one or more amino acid residues, and (c) calculating the ratios of solvent accessibility to solvent exclusion for each of the one or more amino acid residues; whereby a decrease of the ratios for the one or more amino acid residues, relative to the ratios for the same amino acid residues in the same but correctly folded control proteins, indicates the presence and degree of misfolding of the proteins.

2. The method of any one of claim 1, wherein the one or more misfolded proteins comprise transthyretin (TTR) and / or a TTR misfolding surrogate protein.

3. The method of claim 2, wherein the TTR misfolding surrogate protein is TBG, APOB, or APOA1.

4. The method of any one of claims 1-3, wherein the at least one peptide is produced by digestion of the biological sample with a protease.

5. The method of any one of claims 1-4, wherein solvent accessibility and solvent exclusion are determined by differential labeling of the one or more amino acid residues in solvent accessible and solvent excluded states.

6. The method of any one of claims 1-5, wherein the one or more amino acid residues in solvent accessible state is labeled with a first label, and the one or more amino acid residues in solvent excluded state is thereafter labeled with a second label after denaturing the at least one peptide already labeled with the first label.

7. The method of any one of claims 1-6, wherein the one or more amino acid residues are lysine residues.

8. The method of any one of claims 1-7, wherein the biological sample is obtained from a subject having, suspected of having, or suspected of developing ATTR amyloidosis.

9. The method of any one of claims 1-8, wherein the ATTR amyloidosis is wt TTR amyloidosis or V122I TTR amyloidosis.

10. The method of any one of claims 1-9, wherein the biological sample is a plasma sample, a serum sample or a whole blood sample.

11. The method of any one of claims 1-2 and 4-10, wherein the at least one peptide encompasses a destabilized or aggregated region of TTR that is associated with ATTR amyloidosis.

12. The method of any one of claims 1-2 and 4-11, wherein the at least one peptide encompasses TTR β-strand a, b, or c.

13. The method of claim 12, wherein the TTR peptide encompasses one or more amino acid residues selected from Lys35, Lys55, Lys68and Lys146, wherein the amino acid numbering is based on UniProt ID No. P02766.

14. The method of any one of claims 1-12, wherein solvent accessibility and solvent exclusion for the one or more amino acid residues are quantified via mass spectrometry analysis.

15. A method for diagnosing TTR amyloidosis or TTR cardiomyopathy in a subject, comprising (a) obtaining a biological sample from a candidate subject, and (b) quantifying solvent accessibility and solvent exclusion for one or more amino acid residues in one or more misfolded proteins in the biological sample, wherein different molecules of each of the one or more misfolded proteins contain the one or more amino acid residues in both solvent accessible and solvent excluded states; and wherein a decrease of the ratios of solvent accessibility to solvent exclusion for the one or more amino acid residues in the one or more misfolded proteins, relative to the ratios for the same amino acid residues in the same proteins from amyloid-negative control subjects, diagnoses TTR amyloidosis or TTR cardiomyopathy in the candidate subject.

16. The method of claim 15, wherein the quantifying is performed after (a) digesting the biological sample with a protease to generate from each of the one or more misfolded proteins one or more peptides containing the one or more amino acid residues, and (b)differentially labeling the one or more amino acid residues in solvent accessible and solvent excluded states.

17. The method of any one of claims 15-16, wherein the one or more misfolded proteins comprises transthyretin (TTR) and / or a TTR misfolding surrogate protein.

18. The method of any one of claims 15-17, wherein the one or more amino acid residues comprise at least 2, 3, 4 or more different residues in the TTR protein.

19. The method of any one of claims 15-17, wherein the one or more amino acid residues are lysine residues.

20. The method of any one of claims 18, wherein the one or more amino acid residues comprise Lys35, Lys55, Lys68and Lys146of the TTR protein, and wherein the amino acid numbering is based on UniProt ID No. P02766.

21. A method for monitoring treatment effect in a TTR amyloidosis subject undergoing treatment, comprising (a) obtaining a first blood sample from the subject prior to treatment, (b) detecting and quantifying one or more misfolded proteins in the first blood sample, (c) obtaining a second blood sample from the subject during or subsequent to treatment, (d) detecting and quantifying the same one or more misfolded proteins in the second blood sample, and (e) comparing the amount of the one or more misfolded proteins in the two blood samples; wherein misfolded proteins in the blood samples are detected and quantified by (i) generating from each of the blood samples at least one peptide containing one or more amino acid residues in both solvent accessible and solvent excluded states, (ii) quantifying solvent accessibility and solvent exclusion for the one or more amino acid residues for each of the blood samples, and (iii) calculating the ratio of solvent accessibility to solvent exclusion for the one or more amino acid residues; wherein a significant increase in the ratio calculated for the second blood sample over the ratio calculated for the first blood sample indicates a positive treatment effect in the subject.

22. The method of claim 21, wherein the one or more misfolded proteins comprises TTR protein and / or a TTR misfolding surrogate protein.

23. The method of claim 22, wherein the one or more amino acid residues comprises Lys35, Lys55, Lys68and Lys146of the TTR protein, and wherein the amino acid numbering is based on UniProt ID No. P02766.

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