Compositions and Diagnostic Methods Related to Transthyretin

Amyloid Diseases

STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under contract number DK046335 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0002] The present disclosure is in the medical and biomedical field, specifically as it relates to amyloidosis.

BACKGROUND OF THE INVENTION

[0003] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] 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 amyloidoses (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.

[0005] 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. In the case of the familial TTR amyloidoses, caused by the dissociation of TTR tetramers comprising WT and/or mutant TTR subunits, followed by their misfolding and misassembly, the clinical heterogeneity is even more marked. The familial TTR amyloidoses 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 Vall22Ile (present in 4 % of individuals of African descent). Individuals inheriting 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). V30M FAP has high prevalence in endemic areas of Japan and Portugal. Variability in clinical presentation is common and can even occur within the same kindred. Some V30M carriers, e.g., present with marked involvement of other less common organs, such as the eye or the central nervous system and/or the kidneys. For these reasons, TTR amyloidosis patients present first to different clinical specialties, including neurology, cardiology, nephrology, hematology, ophthalmology or gastroenterology. In part 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 amyloidoses - liver transplant and kinetic stabilizer (tafamidis and diflunisal) administration - have proven to be more effective when used early in the course of FAP.

[0006] There is an urgent need in the art for noninvasive, point-of-care, and early diagnostic methods for amyloid diseases such as TTR amyloidoses. The present invention addresses this and other related needs.

SUMMARY OF THE INVENTION

[0007] Various embodiments disclosed herein include a compound comprising: a polypeptide comprising a sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P ₃ and P ₁₀ are each independently any amino acid residue, analog or absent; P2 is a β-branched amino acid residue, analog or absent; P _4i Ρβ and Pg are each independently a β-branched amino acid residue; and Ps _, P7 and P9 are each independently any amino acid residue or analog. In one embodiment, the polypeptide is a synthetic or recombinant peptide. In one embodiment, the polypeptide consists of from 6 to about 20 amino acid residues. In one embodiment, the compound further comprises a diagnostic moiety. In one embodiment, the diagnostic moiety is an alkyne reporter group and/or an alkyl diazirine reactive group. In one embodiment, the diagnostic moiety is conjugated to the polypeptide. In one embodiment, the diagnostic moiety is conjugated to the N-terminal residue of the peptide. In one embodiment, the diagnostic moiety comprises an absorbent, fluorescent or luminescent label moiety. In one embodiment, the diagnostic moiety comprises a fluorophore, a coumarin moiety, or a rhodamine moiety. In one embodiment, P _4i Ρβ and Ps are each Val, P5 is a hydrophobic amino acid residue, P7 is a hydrophobic amino acid residue or His, and P9 is a hydrophobic amino acid residue. In one embodiment, P3 is an uncharged polar amino acid residue or Ala. In one embodiment, Ρ _2ι P _4i Ρβ and Pg are each independently Val or He, P3 is Asn or Ala, P5 is Ala, P7 is His or Ala, and P9 is Phe or Ala. In one embodiment, the polypeptide consists of an amino acid sequence INVAVHVF (SEQ ID NO: 21). In one embodiment, Pi is a hydrophobic amino acid residue or absent, P2 is a β-branched amino acid residue, P3 is an uncharged polar amino acid residue or Ala, and P _lo is Arg, Lys or absent. In one embodiment, Ρ _2ι P _4i Ρβ and Pg are each independently Val or He, Pi is Ala or propargyl glycine, P3 is Asn or Ala, P5 is Ala, P7 is His or Ala, P9 is Phe or Ala, and P _lo is Arg. In one embodiment, the polypeptide consists of an amino acid sequence selected from the group consisting of VAVHVF (SEQ ID NO: 1), AINVAVHVFR (SEQ ID NO: 2), AINAAAHVFR (SEQ ID NO: 3), AINAAVHVFR (SEQ ID NO: 4), AINVAAHVFR (SEQ ID NO: 5), AINVAVHAFR (SEQ ID NO: 6), AANVAVHVFR (SEQ ID NO: 7), AINVAVHVAR (SEQ ID NO: 8), AINVAVAVFR (SEQ ID NO: 9), AIAVAVHVFR (SEQ ID NO: 10), INVAVHVFR (SEQ ID NO: 11), NVAVHVFR (SEQ ID NO: 12), VAVHVFR (SEQ ID NO: 13), AVHVFR (SEQ ID NO: 14), VAVHV (SEQ ID NO: 15), AINVAVHVF (SEQ ID NO: 16), AINVAVHV (SEQ ID NO: 17), AINVAVH (SEQ ID NO: 18), INVAVH (SEQ ID NO: 19), INVAVHV (SEQ ID NO: 20), INVAVHVF(SEQ ID NO: 21), and INVAVHVFR (SEQ ID NO: 22). In one embodiment, the polypeptide consists of an amino acid sequence AINVAVHVFR (SEQ ID NO: 2), and the diagnostic moiety comprises fluorescein-5-carboxamido)hexanoic acid (5-FAM-X). In one embodiment, the polypeptide consists of an amino acid sequence propargyl glycine-INV AVHVFR (SEQ ID NO: 22), and the diagnostic moiety comprises a photoreactive diazirine group and a terminal alkyne group. In one embodiment, provided herein is a composition, comprising the compound of claim 1 and a pharmaceutically acceptable excipient.

[0008] Various embodiments disclosed herein also include a method for diagnosing TTR amyloidosis in a subject, comprising: providing a blood sample of the subject; contacting the blood sample with a compound comprising a polypeptide comprising a sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P ₃ and P ₁₀ are each independently any amino acid residue, analog or absent; P2 is a β-branched amino acid residue, analog or absent; P^ Ρβ and Pg are each independently a β-branched amino acid residue; and Ps _, P7 and P9 are each independently any amino acid residue or analog; and diagnosing TTR amyloidosis if there is binding between a misfolded TTR oligomer in the blood sample and the polypeptide compound. In one embodiment, the blood sample is a plasma sample or a serum sample. In one embodiment, the method further comprises treating the TTR amyloidosis in the subject by liver transplant and/or administering an effective amount of TTR amyloidosis medicine to the subject. In one embodiment, TTR amyloidosis is treated by administering an effective amount of a transthyretin kinetic stabilizer. In one embodiment, the transthyretin kinetic stabilizer is tafamidis and/or diflunisal. In one embodiment, the method further comprises a diagnostic moiety conjugated to the polypeptide compound. In one embodiment, the diagnostic moiety is an alkyne reporter group and/or an alkyl diazirine reactive group. In one embodiment, the diagnostic moiety comprises an absorbent, fluorescent, or luminescent label moiety. In one embodiment, the diagnostic moiety comprises a fluorophore, a coumarin moiety, or a rhodamine moiety. In one embodiment, the specific binding is detected via a fluorescence assay. In one embodiment, the specific binding is detected via photocrosslinking and ELISA assay. In one embodiment, the polypeptide consists of an amino acid sequence selected from the groups VAVHVF (SEQ ID NO: 1), AINV AVHVFR (SEQ ID NO: 2), AINAAAHVFR (SEQ ID NO: 3), AINAAVHVFR (SEQ ID NO: 4), AINVAAHVFR (SEQ ID NO: 5), AINVAVHAFR (SEQ ID NO: 6), AANV AVHVFR (SEQ ID NO: 7), AINVAVHVAR (SEQ ID NO: 8), AINVAVAVFR (SEQ ID NO: 9), AIAV AVHVFR (SEQ ID NO: 10), INV AVHVFR (SEQ ID NO: 11), NVAVHVFR (SEQ ID NO: 12), VAVHVFR (SEQ ID NO: 13), AVHVFR (SEQ ID NO: 14), VAVHV (SEQ ID NO: 15), AINVAVHVF (SEQ ID NO: 16), AINVAVHV (SEQ ID NO: 17), AINVAVH (SEQ ID NO: 18), INVAVH (SEQ ID NO: 19), INVAVHV (SEQ ID NO: 20), INVAVHVF(SEQ ID NO: 21), and INV AVHVFR (SEQ ID NO: 22). [0009] Other embodiments disclosed herein include a method for monitoring treatment of a patient being treated for TTR amyloidosis, comprising: obtaining a first blood sample from the subject prior to treatment, detecting and quantifying misfolded TTR oligomer in the first blood sample by contacting the first blood sample with a compound comprising a polypeptide comprising a sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P ₃ and P ₁₀ are each independently any amino acid residue, analog or absent, P2 is a β-branched amino acid residue, analog or absent, P^ Ρβ and Pg are each independently a β- branched amino acid residue, and Ps _, P7 and P9 are each independently any amino acid residue or analog; obtaining a second blood sample from the subject during or subsequent to treatment, detecting and quantifying misfolded TTR oligomer in the second blood sample by contacting the second blood sample with the polypeptide compound, and monitoring treatment of the patient by comparing the amount of misfolded TTR oligomer in the two blood samples. In one embodiment, the blood sample is a plasma sample or a serum sample. In one embodiment, the method further comprises adjusting the treatment based on the misfolded TTR oligomer in the second blood sample. In one embodiment, the polypeptide in the compound consists of an amino acid sequence selected from the group consisting of VAVHVF (SEQ ID NO: 1), AINVAVHVFR (SEQ ID NO: 2), AINAAAHVFR (SEQ ID NO: 3), AINAAVHVFR (SEQ ID NO: 4), AINVAAHVFR (SEQ ID NO: 5), AINVAVHAFR (SEQ ID NO: 6), AANVAVHVFR (SEQ ID NO: 7), AINVAVHVAR (SEQ ID NO: 8), AINVAVAVFR (SEQ ID NO: 9), AIAVAVHVFR (SEQ ID NO: 10), INVAVHVFR (SEQ ID NO: 11), NVAVHVFR (SEQ ID NO: 12), VAVHVFR (SEQ ID NO: 13), AVHVFR (SEQ ID NO: 14), VAVHV (SEQ ID NO: 15), AINVAVHVF (SEQ ID NO: 16), AINVAVHV (SEQ ID NO: 17), AINVAVH (SEQ ID NO: 18), INVAVH (SEQ ID NO: 19), INVAVHV (SEQ ID NO: 20), INVAVHVF(SEQ ID NO: 21), and INVAVHVFR (SEQ ID NO: 22). In one embodiment, the method further comprises a diagnostic moiety conjugated to the compound. In one embodiment the diagnostic moiety is an alkyne reporter group and/or an alkyl diazirine reactive group.

[0010] A further understanding of the nature and advantages of the present disclosure may be realized by reference to the remaining portions of the specification and claims.

DESCRIPTION OF THE DRAWINGS

[0011] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive. [0012] Figure 1 depicts, in accordance with embodiments herein, that the B β-strand of transthyretin (TTR) labels non-native TTR aggregates. A) Energy landscape associated with the transthyretin aggregation process. B) Strategy for probe design on the hypothesis that peptide fragments of TTR would integrate into defect sites of TTR-derived oligomers and selectively label them. C) TTR tetramer structure (PDB I.D. = 4N85) and sequence, indicating the β-strands (in different colors, labeled from A to H) and segments between the β-strands (in black) that were each individually synthesized. Each peptide was fluorescently labeled at the N-terminus with fluorescein for detection. D) Native PAGE showing incorporation of the B β-strand (probe B-1) into non-native TTR oligomers derived from MTTR. E) Size exclusion chromatography of MTTR oligomers incubated with probe B-1. F) Quantification of incorporation of each candidate TTR-derived peptide probe into MTTR oligomers using size exclusion chromatography. Of all the candidate peptides tested, comprising the entirety of the TTR primary sequence, only the B β-strand peptide significantly labels non-native MTTR oligomers. Inset: AFM image of the MTTR oligomers used in this peptide screen. G) Effect of probe B-1 alanine mutations on incorporation into MTTR oligomers. The amino acids are numbered according to their position in the protein amino acid sequence. Error bars represent mean ± s.d. of at least three separate experiments.

[0013] Figure 2 depicts, in accordance with embodiments herein, that the B β-strand (Bl) is a selective probe for recombinant TTR aggregates within buffer and human plasma. A) Bl labels TTR aggregates and minimally natively folded tetramers. Native gel of Bl incubated with TTR tetramers with the indicated hereditary mutations. B) Effect of alanine mutations on aggregate incorporation as measured by Native PAGE of Bl and 8 variants (SEQ ID NOs: 2-10, respectively). C) Schematic of experimental set-up for panels D-F where fluorescein labeled peptides are incubated within plasma from healthy donors containing MTTR oligomers. Incorporation is measured by Native PAGE or Size Exclusion Chromatography (SEC). D) Native PAGE. E) SEC. F) Effect of alanine mutations on peptide incorporation into MTTR oligomers added into plasma from three healthy donors. The peptides are the same as in panel B (SEQ ID NOs: 2-10, respectively). Incorporation was measured by integrating the peak in the high MW fraction of the SEC chromatogram (shaded in panel E). All errors are ± s.d. *p-value < 0.05.

[0014] Figure 3 depicts, in accordance with embodiments herein, that the B peptide differentiates patients from controls. A) Experimental set-up for panels B-E. B) SEC chromatograms of plasma from three V30M patients and three healthy control incubated with 20 μΜ Bl. C) High MW peak area (150-660 kDa) of V30M symptomatic patients (n=10), healthy donors (n=10), and asymptomatic V30M carriers (n=10). D) The B peptide (Bl) is the only peptide of the TTR β-strands that shows signal in the high MW fraction of the SEC chromatogram from V30M patients (n=3) and healthy donor (n=3). E) Effects of alanine substitution on Bl signal in the high MW fraction from V30M patients (n=3) and healthy donor (n=3). The peptides are the same as in Figure 2 (SEQ ID NOs: 2-10, respectively). All error bars represent ± s.d.

[0015] Figure 4 depicts, in accordance with embodiments herein, that the B peptide differentially labels TTR in patient plasma. A) Experimental set-up for panels B and C. After the incubation of probe B2 (w/ diazirine) or B2C (acetylated control peptide), the plasma mixture is irradiated with 355 nm UV light to activate the photo-crosslinking diazirine functional group. Once photo cross-linked, rhodamine is then conjugated to the alkyne handle, and crosslinked target peptides are visualized by reducing SDS PAGE. B) B2 selectively labels recombinant MTTR oligomers that were doped into healthy donor plasma. C) B2 differentially labels patient plasma (designations beginning with letter "P") compared with control plasma (designations beginning with letter "C"). D) Experimental set-up for panels E and F. After incubation and photo-crosslinking, each sample was fractionated by size exclusion chromatography (SEC, BEH200 Waters Inc., MW range 15-600 kDa). E) Representative SEC chromatogram of a patient and control plasma sample and reducing SDS gel of B-Rhodamine conjugated proteins. Red box: Anti-TTR immunoblot. F) Reducing SDS gel of B-Rhodamine conjugated proteins from the high MW fraction (#3) of 7 V30M FAP patients and 7 healthy donors. G) Experimental set-up for panel H. H) Biotin pulldown and anti-TTR immunoblot of B2 conjugated proteins from the high MW fractions (#3 and #4).

[0016] Figure 5 depicts, in accordance with embodiments herein, that TTR photo- crosslinking by B2 correlates with Bl incorporation into the high MW fractions. A) Band intensity from densitometry of B2 photo-crosslinking of TTR. B) Labeling of TTR in the high MW fraction by B2 photo-crosslinking correlates with the high MW fluorescence signal from the SEC chromatograms (correlation coefficient = 0.85).

[0017] Figure 6 depicts, in accordance with embodiments herein, that Clusterin is a target of B-2. Representative V30M patient and control plasma samples probed with B-2 and an anti-Clusterin antibody (A) and Streptavidin pull-down CuACC click conjugation of Biotin to B-2 after photo-crosslinking (B). [0018] Figure 7 depicts, in accordance with embodiments herein, that probe B-1 detects disease-relevant structures in patient plasma. A) Circulating misfolded transthyretin oligomers in plasma of familial amyloid polyneuropathy patients correlate with early clinical symptoms (r ² =0.5013), and B) decrease upon tafamidis treatment in a subset of patients (upper panel) or remain stable in other patient (lower panel).

[0019] Figure 8 depicts, in accordance with embodiments herein, screen of fragment peptides with TTR derived aggregates or oligomers. Each fragment peptide was incubated overnight with the listed aggregates and incorporation was assessed by size exclusion chromatography. A) MTTR (F87M, L110A) B) C-terminal TTR52-127.

[0020] Figure 9 depicts, in accordance with embodiments herein, that M-TTR oligomers and TTR52-127 actively aggregating more readily incorporate probe B-1 relative to oligomers that have been aged. A) 2 mg/mL M-TTR oligomers that have aged for two months incorporate less peptide than M-TTR oligomers that have aged for 2 weeks. B) 1 mg/mL TTR52-127 aggregates that have aged for 1 day incorporates less B-1 than TTR52-127 that has been aggregating for 3 hrs.

[0021] Figure 10 depicts, in accordance with embodiments herein, that the B-peptide does not incorporate into Αβ1-42 ADDLs. After overnight incubation and size exclusion chromatography, the B-peptide signal shows minimal overlap with the Αβ1-42 signal from the monomer, dimer and trimer species. Overlapping signal is observed in the high MW region (5-6 minutes), although comprises -1% or less of the total B-peptide fluorescent signal.

[0022] Figure 11 depicts, in accordance with embodiments herein, a comparison of binding activities of peptide Bl and several shortened variants (SEQ ID NOs: 2, 11-14, 1 and 15-18, respectively). The data suggest a minimal binding motif of VAVHVF (SEQ ID NO: l). Relative peptide incorporation as measured by Native PAGE. Each B-1 deletion mutant labeled with fluorescein was incubated with 1 week old MTTR oligomers. After fluorescence imaging of the PAGE gel the oligomer band density was normalized to the B-1 sequence (AINVAVHVFR) (SEQ ID NO: 2).

[0023] Figure 12 depicts, in accordance with embodiments herein, linear labeling of M- TTR oligomers in human healthy plasma by probe B-1. Increasing concentrations of MTTR derived oligomers were incubated in healthy human plasma from three different donors and next these oligomers were selectively labeled using B-1 probe (20μΜ). The concentration of B-l peptide in the high molecular weight SEC fraction increased linearly with the increase in MTTR concentration (r ² =0.9215; y=21.8x +35.05); all error bars represent ± s.d.

[0024] Figure 13 depicts, in accordance with embodiments herein, that 4% of healthy controls show some labeling and the presence of cleaved TTR. B-2 rhodamine imaged SDS PAGE gel. Representative example is shown in C5153 where both full length and cleaved TTR are labeled. Controls 5642 and 5609 with minimal labeling are shown for comparison.

[0025] Figure 14 depicts, in accordance with embodiments herein, that the B-peptide does not cross-react with the Anti-TTR antibody (DAKO™, #A0002). MTTR or B-l was spotted onto the membrane and either directly imaged (fluorescein channel) or probed with the polyclonal Anti-TTR antibody from DAKO™.

[0026] Figure 15 depicts, in accordance with embodiments herein, that B-2 does not label the haptoglobin alpha chain. After SEC fractionation of patients and controls as shown in Figure 4E, each fraction was probed with an anti -Haptoglobin antibody. No overlaying signal was observed between anti-Haptoglobin and B-2 or anti-TTR.

[0027] Figure 16 depicts, in accordance with embodiments herein, that probe B-l is selective for nascently formed soluble non-native oligomeric TTR. A) Probe B-l labels A25T TTR derived aggregates and minimally labels natively folded tetramers (TTRtet) as shown in the native PAGE of probe B-l incubated with TTR tetramers with the indicated mutations. B) Probe B-l is most selective for nascently formed MTTR oligomers (~3 days old) as measured by SEC. (Right) Native PAGE showing the molecular weight and labeling intensity differences between 1 -week-old MTTR oligomers and 2-month-old MTTR oligomers. C) Probe B-l-Biotin labeling of salivary gland biopsies of a FAP patient (left panel) and a non-FAP control (right panel). Arrows indicate regions with amyloid fibrils (around the glandular acini in the patient sample), stained by Thioflavin T (ThT; top row) and the anti-TTR antibody (anti-TTR-Alexa594; third row) not labeled by probe B-l-Biotin (B-l- Alexa594; second row). Asterisks indicate regions inside the glandular acini that are labeled by B-l-Biotin in both the patient and the control samples. Scale bars represent 1 mm in length.

[0028] Figure 17 depicts, in accordance with embodiments herein, that probe B-l selectively differentiates FAP patient samples from controls. A) Probe B-l selectively labels the high MW fraction (800-1200 μΐ,, 150-660 kDa) in V30M FAP patients. Experimental setup and representative SEC chromatograms where probe B-l was incubated with the indicated samples for 24 h, before injection onto an SEC column. B) Representative Native PAGE of the samples in panel A. C) High MW peak area (150-660 kDa) of healthy donors (n=62, average age = 43±11 (s.d.)), asymptomatic V30M carriers (n=20, average age = 34±l l(s.d.)), and V30M FAP patients (n=45, average age = 41±14(s.d.)). Comparison by two-tailed t-test. Box plot whiskers represent the 2.5-97.5% range.

[0029] Figure 18 depicts, in accordance with embodiments herein, that probe B-2 labels non-native TTR in patient plasma. A) Chemical structure of probe B-2, containing a photoactivatable crosslinker (diazirine) and reporter (alkyne) functional group, and probe B- 2C, lacking the diazirine. B) Schematic of the photo-crosslinking approach used to generate panels C and D, whereby B-2 is incubated with patient plasma, photocrosslinked via the diazirine and then visualized by reducing SDS PAGE analysis after rhodamine conjugation to the alkyne handle. C) Probe B-2 selectively and covalently labels recombinant MTTR oligomers that were added to healthy donor plasma. TTR labeling by B-2 is indicated by the magenta box. D) Probe B-2 differentially covalently labels TTR in patient plasma, but not in control plasma because the probe does not bind to native TTR. TTR labeling by B-2 is indicated by the magenta box. E) Experimental set-up for panels F, G and H. After incubation and photo-crosslinking, each sample was fractionated by size exclusion chromatography (SEC) before conjugation of rhodamine or biotin to the alkyne handle (the high [Cu] in this reaction denatures the proteome, thus non-denaturing SEC is done first). F) Representative SEC chromatogram of a patient and control plasma sample. Reducing SDS PAGE of probe B2-rhodamine conjugated proteins (middle panel), TTR labeling by B-2 is indicated by the magenta box. Anti-TTR immunoblot is indicated by the dark green box (bottom panel). G) SDS-PAGE band densitometry quantification of probe B-2 TTR labeling in the high MW SEC fractions (fractions 3 & 4, see fig. 31 for experimental design schematic and additional representative data) from healthy donors (n=30), V30M asymptomatic mutation carriers (n=13), and V30M FAP patients (n=43). p-values were calculated by two-tailed t-test. Box plot whiskers represent the 2.5-97.5% range. H) Biotin pulldown and anti-TTR immunoblot of probe B2-conjugated proteins from the high MW fraction #4.

[0030] Figure 19 depicts, in accordance with embodiments herein, that targeted mass spectrometry confirms non-native TTR as a target of the B-peptide. A) Experimental scheme whereby either probe B-2 or a probe B-2 mutant (V28A, B-2-Mut) that does not incorporate into non-native TTR, is incubated with either patient or control plasma. After photocrosslinking, biotin is conjugated to the alkyne handle and the probe B-2-biotin conjugates enriched with streptavidin. After trypsin digestion, the trypsin fragments are labeled with one of six TMT tags, then combined and subjected to MudPIT LC MS/MS analysis. B) Rhodamine fluorescence labeling of non-native TTR oligomers from a V30M FAP patient incubated with probe B-2 compared to probe B-2-Mut (magenta box). C) Venn diagram showing the overlap of proteins identified from healthy blood donors, V30M asymptomatic mutation carriers, and V30M FAP patients. D) Intensity of TTR peptide signal from plasma of three FAP patients treated with either probe B-2 or probe B-2-Mut. E) Volcano plot of identified proteins quantified by TMT isobaric mass tags shown as a ratio of the intensity in the probe B-2 TMT channel to that of the corresponding probe B-2-Mut TMT channel. Enrichment ratios are shown as an average (n=3) and p-values were calculated from the log2 transformed values using a two-tailed t-test. F) Probe B-2 to probe B-2-Mut ratios for a select number of proteins previously identified in TTR amyloid deposits, p-values were calculated using a two-tailed t-test (* < 0.05, ** < 0.01, *** < 0.001).

[0031] Figure 20 depicts, in accordance with embodiments herein, labeling of high molecular weight non-native TTR oligomers decreases V30M FAP patients treated with tafamidis or liver transplantation. A) Representative probe B-2 rhodamine gel images from the high MW SEC fraction from 15 Portuguese FAP patients whose blood was taken prior to treatment and after 12 months treatment with the kinetic stabilizer tafamidis. The NIS-LL score for each patient and the change after 12 months is listed below each gel. Of the 15 patients analyzed, 13 either decreased or remain unchanged and 2 patients showed an increase in non-native TTR labeling. B) Quantification of probe B-2 non-native TTR labeling by band densitometry (left) and fold change after 12 months of treatment (right). In the 15 patients analyzed, the non-native TTR signal decreased 1.8 fold on average from 6.7±0.9 to 3.7±0.5 (mean±s.e.m). p-value was calculated by a two-tailed unpaired t-test comparing the Pre-Tafamidis and 12 mo time points. C) Non-native TTR labeling by B-2 of 7 V30M FAP patients (Japan) whom underwent liver transplantation (0.8±0.3; mean±s.e.m).

[0032] Figure 21 depicts, in accordance with embodiments herein, non-native TTR is detected in additional ATTR mutations and not detected in cardiomyopathy-associated genotypes (V122I and WT). (Top) Experimental schematic. (Bottom) Non-native TTR levels as detected by B-2 photocros slinking and SDS-PAGE. p-values were calculated using a two- tailed unpaired t-test. Comparisons in which the p-value is greater than 0.05 are not shown. V30M FAP data from Fig. 4G is presented for comparison.

[0033] Figure 22 depicts, in accordance with embodiments herein, that the B β-strand of TTR labels TTR50-127 oligomers. (A) AFM image of TTR50-127 oligomers that were used to assay incorporation. (B) Quantification of incorporation of each candidate TTR-derived peptide probe into TTR50-127 oligomers using size exclusion chromatography. [0034] Figure 23 depicts, in accordance with embodiments herein, determination of probe stoichiometry in MTTR Oligomers. A) Standard curve of B-1 in 6M Guanidine-HCl. B) Method for determination of fluorescence quenching and stoichiometry of B-1 bound to recombinant oligomers. C) Representative emission scan showing -10 fold quenching of the fluorescein fluorescence intensity when B-1 is bound to the MTTR oligomers.

[0035] Figure 24 depicts, in accordance with embodiments herein, additional microscopy images showing that the B-peptide does not label V30M FAP derived TTR amyloid in salivary gland biopsies. A) V30M FAP salivary gland biopsy images. B) Healthy control salivary gland biopsy images.

[0036] Figure 25 depicts, in accordance with embodiments herein, that probe B-1 linearly labels MTTR oligomers in human healthy plasma. Increasing concentrations of MTTR derived oligomers were incubated in healthy human plasma from three different donors and next these oligomers were selectively labeled using B-1 probe. The concentration of B-1 in the high molecular weight SEC fraction increased linearly with the increase in non-native TTR concentration (r2=0.9215; y=21.8x +35.05); all error bars represent ± s.d.

[0037] Figure 26 depicts, in accordance with embodiments herein, that a similar B-1 probe Structure Activity Relationship (SAR) is found when MTTR oligomers are incubated in healthy plasma. MTTR oligomers were incubated in plasma of three healthy donors and the incorporation of the B-1 alanine mutants was measured by size exclusion chromatography (see Fig. 1G for similar results in buffer). Each bar represents the mean of the three plasma samples for a specified mutant; all error bars represent ± s.d.

[0038] Figure 27 depicts, in accordance with embodiments herein, that labeling of the high MW SEC fraction in FAP patients by disulfoCy5-B is fluorophore independent.

[0039] Figure 28 depicts, in accordance with embodiments herein, that alanine substitutions of probe B-1 have identical effects on incorporation of peptide into the HMW fraction of FAP V30M patient plasma. The alanine mutants incorporation was measured in three separate patient plasma samples and three healthy control samples using the size exclusion chromatography method. For comparison, B-1 data from all patients and controls analyzed with probe B-1 is shown (patients: n=45, controls: n=62). Each bar represents the mean of the three plasma samples for a specified mutant; all error bars represent ± s.d.

[0040] Figure 29 depicts, in accordance with embodiments herein, that B-1 is the only peptide of the TTR β-strands that incorporates into the high MW fraction of patient plasma. The 8 β-strands were incubated with patient plasma (n=3 for strands A, and C-H; n=45 for B- 1) and peptide incorporation was measured by size exclusion chromatography. Each bar represents the mean of the three plasma samples for a specified mutant; all error bars represent ± s.d.

[0041] Figure 30 depicts, in accordance with embodiments herein, that Diazirine- containing probe B-2 selectively labels oligomeric TTR. A) Probe B-2 selectively labels non- native oligomeric TTR but not mutant or WT tetramers. B) Probe B-2 labels oligomeric TTR but not the folded monomer.

[0042] Figure 31 depicts, in accordance with embodiments herein, the schematic of B-2 non-native TTR gel quantification method and representative data. After incubation with probe B-2, the samples were photocrosslinked, separated by SEC using the Agilent Bio SEC- 3 4.6 x 300 mm column and N3-Rhodamine was then clicked onto the B-2 alkyne handle. The samples were then analyzed by reducing SDS PAGE and the bands were quantified by densitometry and then normalized to the total protein present. To compare samples between different gels, each gel was loaded with a standard curve of retroaldolase labeled quantitatively with a single alkyne as described in the Methods. The gel above depicts 5 plasma samples from V30M asymptomatic carriers (5 unique individuals) and 5 V30M FAP patients (5 unique individuals).

[0043] Figure 32 depicts, in accordance with embodiments herein, that probe B-1 does not cross-react with the anti-TTR antibody. MTTR or probe B-1 was spotted onto the nitrocellulose membrane and either directly imaged (fluorescein channel) or probed with the polyclonal Anti-TTR antibody.

[0044] Figure 33 depicts, in accordance with embodiments herein, correlation of the spectral counts (MSI spectra) of the identified protein targets in the B-2/B-2-Mut treated samples from V30M FAP patients (average of 3 patients) with plasma concentration. Approximate human plasma concentrations were obtained from the Plasma Proteome Database (accessed October 1, 2016).

[0045] Figure 34 depicts, in accordance with embodiments herein, validation of N- terminally cleaved non-native TTR as a target of the B-peptide in V30M FAP patient plasma. A) N-terminally cleaved non-native TTR elutes in the high MW fraction. SEC fractionation of patient #11 (representative example) with B-2 and visualized by rhodamine conjugation to the alkyne handle (magenta box) or by anti-TTR western blot using a C-terminal specific antibody (dark-green box). Patient sample numbers correspond to the same patient sample labels that were shown in Fig. 20. B) Western blot of plasma samples analyzed by SDS PAGE and probed with a monoclonal C-terminal specific anti-TTR antibody. C) N-terminally cleaved TTR is not generated by a protease during the peptide incubation period. Identical results were observed with or without the addition of a protease inhibitor cocktail.

DETAILED DESCRIPTION OF THE INVENTION

[0046] All references, publications, and patents cited herein are incorporated by reference in their entirety as though they are fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

[0047] As described herein, and in accordance with the various embodiments disclosesd herein, the inventors have developed novel peptide-based probes that allow detection of misfolded transthyretin oligomers in the plasma of patients with hereditary transthyretin amyloid polyneuropathy and these probes reveal that the levels of these oligomers are lowered upon treatment with disease-modifying therapies.

[0048] Increasing evidence supports the hypothesis that soluble misfolded protein assemblies contribute to the degeneration of post-mitotic tissue in amyloid diseases. However, there is a dearth of reliable non-antibody based probes for selectively detecting oligomeric aggregate structure(s) circulating in plasma or deposited in tissues, making it difficult to scrutinize such hypotheses in patients. Hence, understanding the structure- proteotoxicity relationships driving amyloid diseases remains challenging, hampering the development of early diagnostic strategies. Herein, the inventors have disclosed peptide- based probes that selectively label misfolded transthyretin (TTR) oligomers circulating in the plasma of TTR hereditary amyloidosis patients with a predominant neuropathic phenotype. These probes reveal that misfolded TTR oligomer levels are much lower in healthy controls, in asymptomatic carriers of mutations linked to amyloid polyneuropathy, and in patients with TTR-associated cardiomyopathies. Notably, oligomer levels decrease in TTR amyloid polyneuropathy patients treated with disease-modifying therapies (Tafamidis or liver transplant-mediated gene therapy). Quantification of plasma oligomer levels by such peptide probes could become an early diagnostic strategy, a response-to-therapy biomarker, and a useful tool for understanding structure-proteotoxicity relationships in the TTR amyloidoses.

[0049] In one embodiment disclosed herein is a compound comprising a polypeptide comprising a sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P ₃ and P ₁₀ are each independently any amino acid residue, analog or absent; P2 is a β-branched amino acid residue, analog or absent; P _4i Ρβ and Pg are each independently a β- branched amino acid residue; and Ps _, P7 and P9 are each independently any amino acid residue or analog. In one embodiment, the polypeptide is a synthetic or recombinant peptide. In one embodiment, the polypeptide consists of from 6 to about 20 amino acid residues. In one embodiment, the compound further comprises a diagnostic moiety. In one embodiment, the diagnostic moiety is an alkyne reporter group and/or an alkyl diazirine reactive group. In one embodiment, the diagnostic moiety is conjugated to the polypeptide. In one embodiment, the diagnostic moiety is conjugated to the N-terminal residue of the peptide. In one embodiment, the diagnostic moiety comprises an absorbent, fluorescent or luminescent label moiety. In one embodiment, the diagnostic moiety comprises a fluorophore, a coumarin moiety, or a rhodamine moiety. In one embodiment, P _4i Ρβ and Ps are each Val, P5 is a hydrophobic amino acid residue, P7 is a hydrophobic amino acid residue or His, and P9 is a hydrophobic amino acid residue. In one embodiment, P3 is an uncharged polar amino acid residue or Ala. In one embodiment, Ρ _2ι P _4i Ρβ and Pg are each independently Val or He, P3 is Asn or Ala, P5 is Ala, P7 is His or Ala, and P9 is Phe or Ala. In one embodiment, the polypeptide consists of an amino acid sequence INVAVHVF (SEQ ID NO: 21). In one embodiment, Pi is a hydrophobic amino acid residue or absent, P2 is a β-branched amino acid residue, P3 is an uncharged polar amino acid residue or Ala, and P _lo is Arg, Lys or absent. In one embodiment, Ρ _2ι Ρ _4ι Ρβ and Pg are each independently Val or He, Pi is Ala or propargyl glycine, P3 is Asn or Ala, P5 is Ala, P7 is His or Ala, P9 is Phe or Ala, and P _lo is Arg. In one embodiment, the polypeptide consists of an amino acid sequence selected from the group consisting of VAVHVF (SEQ ID NO: 1), AINVAVHVFR (SEQ ID NO: 2), AINAAAHVFR (SEQ ID NO: 3), AINAAVHVFR (SEQ ID NO: 4), AINVAAHVFR (SEQ ID NO: 5), AINVAVHAFR (SEQ ID NO: 6), AANVAVHVFR (SEQ ID NO: 7), AINVAVHVAR (SEQ ID NO: 8), AINVAVAVFR (SEQ ID NO: 9), AIAVAVHVFR (SEQ ID NO: 10), INVAVHVFR (SEQ ID NO: 11), NVAVHVFR (SEQ ID NO: 12), VAVHVFR (SEQ ID NO: 13), AVHVFR (SEQ ID NO: 14), VAVHV (SEQ ID NO: 15), AINVAVHVF (SEQ ID NO: 16), AINVAVHV (SEQ ID NO: 17), AINVAVH (SEQ ID NO: 18), INVAVH (SEQ ID NO: 19), INVAVHV (SEQ ID NO: 20), INVAVHVF(SEQ ID NO: 21), and INVAVHVFR (SEQ ID NO: 22). In one embodiment, the polypeptide consists of an amino acid sequence AINVAVHVFR (SEQ ID NO: 2), and the diagnostic moiety comprises 6-(fluorescein-5-carboxamido)hexanoic acid (5- FAM-X). In one embodiment, the polypeptide consists of an amino acid sequence propargyl glycine-INVAVHVFR (SEQ ID NO: 22), and the diagnostic moiety comprises a photoreactive diazirine group and a terminal alkyne group.

[0050] In another embodiment, disclosed herein is a method for diagnosing TTR amyloidosis in a subject, comprising: providing a blood sample of the subject; contacting the blood sample with a compound comprising a polypeptide comprising a sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P ₃ and P ₁₀ are each independently any amino acid residue, analog or absent; P2 is a β-branched amino acid residue, analog or absent; P^ Ρβ and Pg are each independently a β-branched amino acid residue; and Ps _, P7 and P9 are each independently any amino acid residue or analog; and diagnosing TTR amyloidosis if there is binding between a misfolded TTR oligomer in the blood sample and the polypeptide compound. In one embodiment, the blood sample is a plasma sample or a serum sample. In one embodiment, the method further comprises treating the TTR amyloidosis in the subject by administering an effective amount of TTR amyloidosis medicine to the subject. In one embodiment, TTR amyloidosis is treated by administering an effective amount of a transthyretin kinetic stabilizer. In one embodiment, the transthyretin kinetic stabilizer is tafamidis. In one embodiment, the method further comprises a diagnostic moiety conjugated to the polypeptide compound. In one embodiment, the diagnostic moiety is an alkyne reporter group and/or an alkyl diazirine reactive group. In one embodiment, the diagnostic moiety comprises an absorbent, fluorescent, or luminescent label moiety. In one embodiment, the diagnostic moiety comprises a fluorophore, a coumarin moiety, or a rhodamine moiety. In one embodiment, the specific binding is detected via a fluorescence assay. In one embodiment, the specific binding is detected via photocrosslinking and ELISA assay. In one embodiment, the polypeptide consists of an amino acid sequence selected from the groups VAVHVF (SEQ ID NO: 1), AINVAVHVFR (SEQ ID NO: 2), AINAAAHVFR (SEQ ID NO: 3), AINAAVHVFR (SEQ ID NO: 4), AINVAAHVFR (SEQ ID NO: 5), AINVAVHAFR (SEQ ID NO: 6), AANVAVHVFR (SEQ ID NO: 7), AINVAVHVAR (SEQ ID NO: 8), AINVAVAVFR (SEQ ID NO: 9), AIAVAVHVFR (SEQ ID NO: 10), INVAVHVFR (SEQ ID NO: 11), NVAVHVFR (SEQ ID NO: 12), VAVHVFR (SEQ ID NO: 13), AVHVFR (SEQ ID NO: 14), VAVHV (SEQ ID NO: 15), AINVAVHVF (SEQ ID NO: 16), AINVAVHV (SEQ ID NO: 17), AINVAVH (SEQ ID NO: 18), INVAVH (SEQ ID NO: 19), INVAVHV (SEQ ID NO: 20), INVAVHVF(SEQ ID NO: 21), and INVAVHVFR (SEQ ID NO: 22).

[0051] In another embodiment disclosed herein is a method for monitoring treatment of a patient being treated for TTR amyloidosis, comprising: obtaining a first blood sample from the subject prior to treatment, detecting and quantifying misfolded TTR oligomer in the first blood sample by contacting the first blood sample with a compound comprising a polypeptide comprising a sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P ₃ and P ₁₀ are each independently any amino acid residue, analog or absent, P2 is a β-branched amino acid residue, analog or absent, P^ Ρβ and Pg are each independently a β- branched amino acid residue, and Ps _, P7 and P9 are each independently any amino acid residue or analog; obtaining a second blood sample from the subject during or subsequent to treatment, detecting and quantifying misfolded TTR oligomer in the second blood sample by contacting the second blood sample with the polypeptide compound, and monitoring treatment of the patient by comparing the amount of misfolded TTR oligomer in the two blood samples. In one embodiment, the blood sample is a plasma sample or a serum sample. In one embodiment, the method further comprises adjusting the treatment based on the misfolded TTR oligomer in the second blood sample. In one embodiment, the polypeptide in the compound consists of an amino acid sequence selected from the group consisting of VAVHVF (SEQ ID NO: 1), AINVAVHVFR (SEQ ID NO: 2), AINAAAHVFR (SEQ ID NO: 3), AINAAVHVFR (SEQ ID NO: 4), AINVAAHVFR (SEQ ID NO: 5), AINVAVHAFR (SEQ ID NO: 6), AANVAVHVFR (SEQ ID NO: 7), AINVAVHVAR (SEQ ID NO: 8), AINVAVAVFR (SEQ ID NO: 9), AIAVAVHVFR (SEQ ID NO: 10), INVAVHVFR (SEQ ID NO: 11), NVAVHVFR (SEQ ID NO: 12), VAVHVFR (SEQ ID NO: 13), AVHVFR (SEQ ID NO: 14), VAVHV (SEQ ID NO: 15), AINVAVHVF (SEQ ID NO: 16), AINVAVHV (SEQ ID NO: 17), AINVAVH (SEQ ID NO: 18), INVAVH (SEQ ID NO: 19), INVAVHV (SEQ ID NO: 20), INVAVHVF(SEQ ID NO: 21), and INVAVHVFR (SEQ ID NO: 22). In one embodiment, the method further comprises a diagnostic moiety conjugated to the compound. In one embodiment the diagnostic moiety is an alkyne reporter group and/or an alkyl diazirine reactive group.

[0052] In one embodiment, provided herein is a composition, comprising a compound comprising a polypeptide having the sequence P1P2P3P4P5P6P7P8P9P10 or a pharmaceutically acceptable salt thereof, wherein Pi, P ₃ and P _lo are each independently any amino acid residue, analog or absent; P2 is a β-branched amino acid residue, analog or absent; P^ Ρβ and Pg are each independently a β-branched amino acid residue; and Ps _, P7 and P9 are each independently any amino acid residue or analog; and a pharmaceutically acceptable excipient. "Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0053] In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. "Route of administration" may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral. "Parenteral" refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.

[0054] The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0055] The pharmaceutical compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0056] The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

[0057] The pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 21st edition, Williams & Wilkins PA, USA) (2005).

[0058] Typical dosages of an effective composition can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activity. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of the relevant primary cultured cells or histocultured tissue sample, such as biopsied malignant tumors, or the responses observed in the appropriate animal models.

I. Overview

[0059] The present disclosure is predicated in part on the studies undertaken by the present inventors to investigate aggregate structure-proteotoxicity relationships in amyloid diseases. The studies were intended to better understand the spectrum of aggregate structures that exist in patients, and to develop probes for aggregate structures that may drive proteotoxicity. As detailed herein, the inventors aimed to develop probes that can selectively and ideally specifically detect soluble non-native TTR structures that form in addition to amyloid fibrils. 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 (T ₄ ) and holo-retinol binding protein (RBP). TTR is one of greater than 20 nonhomologous amyloidogenic 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 misassembly into amyloid that causes wildtype TTR amyloidsis (WT-ATTR). Dissociation and misfolding of one of more than 120 TTR variants results in hereditary ATTR amyloidoses, including familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC).

[0060] TTR amyloid diseases (TTR amyloidoses or ATTR amyloidoses) 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.

[0061] The inventors synthesized peptide-based probes that selectively integrate into the structure(s) of the non-native TTR oligomers that can be prepared in vitro and integrate into apparently similar structures that circulate in TTR polyneuropathy and cardiomyopathy patient blood. This approach of quantifying misfolded TTR oligomers in patient plasma could be useful not only for aiding physicians in point-of-care diagnosis and in following the response to particular therapies, but also for basic science purposes in terms of understanding the aggregate structure-proteotoxicity relationships driving the TTR amyloidoses.

[0062] The methods described herein enables detection of non-native TTR oligomers in patients suffering from TTR amyloidoses - both polyneuropathy and cardiomyopathy phenotypes. An important aspect of the probes described herein is that, unlike other probes used for diagnostic purposes (i.e., Congo Red or thioflavin T), these probes preferentially recognize nascently misfolded and actively aggregating TTR, making them suitable for detection of the earliest possible misfolding events that lead to TTR related amyloid diseases. The same approach is generalizable to other systemic amyloid diseases, such as those caused by aggregating antibody light chains (AL and amyloidosis in multiple myeloma), as well as other amyloidogenic proteins such as lysosozyme and β-2-microglobulin. [0063] The TTR related systemic amyloid disease class and the probes disclosed herein provide the initial tools to understand the tissue tropism of the amyloid diseases. For example, why is the peripheral nervous system affected in V30M-FAP, versus the heart in VI 221 related cardiomyopathies? The probes developed here will certainly assist future studies directed at elucidating the structure-proteotoxicity relationship driving loss of postmitotic tissue, which is currently a major gap in understanding amyloid diseases mechanisms. For example, a specific probe described herein, probe B-2, can detect circulating TTR that is fragmented, which heretofore has only been detected in amyloid tissue biopsies, and may explain particular aspects of the tissue tropism associated with these diseases. It is possible that particular variants including the WT protein are or become susceptible to proteolysis, which leads to non-native TTR that preferentially targets the heart.

[0064] In addition, it was found that the non-native oligomeric TTR that probes B-l and B-2 detect in patient blood is not without other protein partners. The inventors identified at least one oligomeric TTR interacting protein partner, the extracellular chaperone Clusterin. The expression and presence of these partner proteins could be together considered in addition to the detection of non-native TTR to assess disease status. Finally, the peptidomimetic probes enable numerous diagnostic applications described herein. For example, both the probe B-l fluorescence assay and the probe B-2 photo-crosslinking and SDS-PAGE gel assay described in the Examples below can be applied to other amyloid diseases to detect the onset of these diseases at the earliest possible time-point.

[0065] In accordance with these discoveries, the present disclosure provides novel peptides or peptidomimetic probes that can be used in diagnosing distinct TTR amyloid diseases or monitoring disease status. Also provided in the instant disclosure are various diagnostic uses and disease monitoring applications of such probes.

II. Definitions

[0066] 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 of Microbiology 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 (4 ed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0067] 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.

[0068] As used herein, the terms "peptide" and "polypeptide," are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length unless otherwise defined. Peptides and polypeptides are composed of linearly arranged amino acids linked by peptide bonds, and may be produced biologically and isolated from the natural environment, produced using recombinant technology, or produced synthetically typically using naturally occurring amino acids. In some aspects, the polypeptide is a "modified polypeptide" comprising non-naturally occurring amino acids. In some aspects, the polypeptides comprise a combination of naturally occurring and non-naturally occurring amino acids, and in some embodiments, the peptides comprise only non-naturally occurring amino acids. The term peptide is contemplated to encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to, N-terminus modification, C-terminus modification, peptide bond modification, backbone modification, and/or side chain modification.

[0069] 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., an 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 present disclosure may encompass derivative or analogs which have be modified with non-naturally coding amino acids.

[0070] 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 (He), 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 (Gin), 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).

[0071] 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 amyloidoses, 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 as rheumatoid 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.

[0072] As used herein, a "branched-chain amino acid" is an amino acid having aliphatic side-chains with a branch (a central carbon atom bound to three or more carbon atoms). Among the proteinogenic amino acids, there are two β-branched amino acids (isoleucine and valine) and one γ-branched amino acid (leucine). In addition, non-proteinogenic branched- chain amino acids include norvaline and 2-aminoisobutyric acid. In some embodiments, the peptide probes of the instant disclosure may include non-proteinogenic branched-chain amino acids. [0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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).

[0078] 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.

[0079] 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.

[0080] 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. Nat'l. 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.

[0081] Other than percentage of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded 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.

[0082] 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 transthyretin (TTR) derived peptide or variant, the diagnostic moiety or label moiety does not encompass peptide sequences that are naturally present in the TTR protein. Typically, the diagnostic moiety or label moiety contemplated in the instant disclosure 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.

[0083] 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 misassembly of TTR monomers. Unlike normal circulating TTR which are tetrameric, these TTR aggregates results from misfolding and misassembly of monomeric TTR molecules released by dissociation of the normal tetrameric TTR.

[0084] The term "photoactivatable crosslinker" is used herein to indicate a reactive functional group capable of becoming covalently bound to another molecule upon irradiation by light, preferably ultraviolet light. A diazirine ring is an example photoactivatable crosslinker that can be employed in the instant disclosure, and it typically involves the generation of a carbene. A diazirine functional group has advantages in that it will only result in minimal structural changes within the target molecule to which it binds. [0085] The term "terminal alkyne group" refers to reactive species in a number of important chemical reactions such as the 1,3-dipolar cycloadditionl between azides and alkynes to give 1,2,3-triazoles (click-chemistry), the Sonogashira reaction and its forerunner, the Stephens-Castro reaction. Furthermore, alkynes can undergo the Vollhardt cyclization, alkyne trimerization to form aromatic compounds, or can act as dienophiles in Diels-Alder reactions. A number of protecting groups have been developed for alkyne chemistry such as trialkylsilyl, benzyl- or phenyl-substituted alkylsilyl groups and propargylic alcohols. In some preferred embodiments, a terminal alkyne group used in the present disclosure is capable of being covalently linked in a chemical reaction with a molecule containing an azide. The terminal alkyne or azide can serve as a non-native and non-perturbing bioorthogonal chemical handle that can be derivatized employing a chemistry that is known as click chemistry.

[0086] The term "photolysis" is meant to indicate the light induced activation of a photoactivatable group (e.g., a diazirine functional group) resulting in a reactive species (e.g., carbene) that can form a covalent linkage with a molecule in close proximity. Once the photoactivatable group is covalently attached to the target molecule (e.g., a TTR derived peptide), the latter can be identified or visualized by methods well known in the art. The process of attaching a target molecule to a terminal alkyne or azide group is described in Rostovtsev et al., Angew. Chem. Int. Ed., 2002, and Tornoe et al, J. Org. Chem. 2002.

[0087] 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.).

[0088] As used herein, the term "peptide mimetic" or "peptidomimetic" refers to a derivative compound of a reference peptide (e.g., a TTR derived probe polypeptide 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.

[0089] 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. [0090] As used herein, the term "variant" refers to a molecule (e.g., a polypeptide or peptide) 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. Probe peptides or variants for incorporation into misfolded TTR oligomers

[0091] In one embodiment, the instant disclosure provides synthetic or recombinantly produced peptides that are capable of specifically binding to soluble non-native TTR oligomers. As exemplified herein, these probe peptides or peptide probes can selectively integrate into both the structure of non-native TTR oligomers prepared in vitro and similar structures found in the blood of TTR amyloidosis patients. The peptide probes disclosed herein typically contain from 6 amino acid residues to about 20 amino acid residues. In various embodiments, the peptide probes can contain 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues. In some preferred embodiments, the peptides contain from 6 amino acid residues to about 12 amino acid residues. As exemplified herein, some of these peptides contain about 8 amino acid residues. Some other peptides contain about 9 amino acid residues. Some other peptides contain about 10 amino acid residues.

[0092] In some embodiments, peptides of the present disclosure also contain a 6-aa sequence motif P4P5P6P7P8P9. In this sequence, residues Ρ _4ι Ρβ and Pg are each independently a branched amino acid residue such as β-branched residues, and the other residues each independently may be any amino acid residue or analog. In some of these embodiments, residue P5 is a hydrophobic amino acid residue, residue P7 is a hydrophobic amino acid residue, His or Lys, and residue P9 is a hydrophobic amino acid residue. In some embodiments, residues P _4i Ρβ and Pg are each independently Val or He, residue P5 is Ala, residue P7 is His or Ala, and residue P9 is an aromatic amino acid residue or Ala. In some peptides, residues P _4i Ρβ and Pg are each Val, residue P5 is Ala, residue P7 is His or Ala, and residue P9 is Phe, Tyr or Ala. In some embodiments, the peptides contain a 7-aa sequence motif. Relative to the 6-aa sequence motif, the motif additionally contains an extra β- branched amino acid residue at its C-terminus. In some peptides, the 6-aa or 7-aa sequence motif is VAVHVF (SEQ ID NO: 1), INVAVH (SEQ ID NO: 19), or INVAVHV (SEQ ID NO: 20). As demonstrated herein, a number of peptides of varying length that harbor one of these sequence motifs are able to bind to misfolded TTR oligomers. These include 6-aa peptide VAVHVF (SEQ ID NO: 1), 7-aa peptide VAVHVFR (SEQ ID NO: 13), 8-aa peptides NVAVHVFR (SEQ ID NO: 12) and AINVAVHV (SEQ ID NO: 17).

[0093] In some embodiments, the peptides contain an 8-aa sequence motif P2P ₃ P4P5P ₆ P7- PgP i. Relative to the 6-aa motif, residue P2 in this 8-aa sequence is a branched amino acid residue such as β-branched residue Val or He, and residue P3 is any amino acid residue or analog. In some peptides, residue P3 is an uncharged polar amino acid residue or Ala. In some of these peptides, residues P _2i P _4i Ρβ and Pg are each independently Val or He, residue P3 is Asn or Ala, residue P5 is Ala, residue P7 is His or Ala, and residue P9 is Phe or Ala. Examples of such peptides that have shown activities in binding to misfolded TTR oligomer include, e.g., 8-aa peptide INVAVHVF (SEQ ID NO: 21), and 9-aa peptides INVAVHVFR (SEQ ID NO: 11) and AINVAVHVF (SEQ ID NO: 16).

[0094] In some embodiments, the peptides contain a sequence motif P1P2P3P4P5P6P7- P ₈ P ₉ P1 ₀ . Relative to the 6-aa motif, residue Pi in this sequence motif can be any amino acid residue, analog or absent, residue P2 is a β-branched amino acid residue, residue P3 is any amino acid residue or analog, and residue P ₁₀ is any amino acid residue, analog or absent. In some peptides, residue Pi is a hydrophobic amino acid residue or absent, residue P2 is a β- branched amino acid residue, residue P3 is an uncharged polar amino acid residue or Ala, and residue P _lo is Arg, Lys or absent. In some of these peptides, residues Ρ _2ι Ρ _4ι Ρβ and Pg are each independently Val or He, residue Pi is Ala, residue P3 is Asn or Ala, residue P5 is Ala, residue P7 is His or Ala, residue P9 is Phe or Ala, and residue P _lo is Arg. Exemplary peptides harboring such a sequence motif that have demonstrated binding activity for misfolded TTR oligomer include AINVAVHVFR (SEQ ID NO: 2), propargyl glycine-INVAVHVFR (SEQ ID NO: 22), AIAVAVHVFR (SEQ ID NO: 10), AINVAVAVFR (SEQ ID NO: 9), and AINVAVHVAR (SEQ ID NO: 8).

[0095] In a related aspect, the present disclosure provides peptide probe compounds that contain a synthetic or recombinant peptide and a diagnostic moiety. For example, in one embodiment, the present disclosure provides modifying the peptides of SEQ ID Nos: 1-22 by incorporating a diazirine functional group and an alkyne handle. One non-limiting example of such a probe is probe B-2 as illustrated below. In one embodiment, it may be advantageous to use probe B-2 over probe B-l because after photocrosslinking, probe B-2 is covalently attached to the proteins it initially binds to, thus denaturing and reducing SDS PAGE can be used to identify the proteins bound by probe B-2, including initially aggregated non-native TTR (Fig. 18B). In contrast, non-denaturing separation methods like SEC must be used to preserve the non-covalent probe B-l interactions. NVAVHVFR

Probe B-2

[0096] In one embodiment, probe B-2 displays a high degree of conformational selectivity for non-native TTR oligomers, as does probe B-l . Control experiments with peptide probe B- 2C, lacking the diazirine functional group, exhibited minimal TTR labeling, indicating that labeling is dependent on the presence of the diazirine cross-linker, facilitating denaturing SDS-PAGE separation and analysis.

[0097] In one embodiment, an important advantage of the covalent peptide-based probe B- 2 introduced herein is that, unlike other amyloid-selective probes used for diagnostic purposes (e.g., Congo Red or thioflavin T), these probes preferentially recognize soluble, misfolded and actively aggregating TTR oligomers that adopt a non-amyloid conformation, rendering these probes suitable for detection of early misfolding events that may lead to degenerative phenotypes characteristic of the TTR amyloidoses. Importantly, these probes detect non-native TTR in neuropathic TTR amyloidosis patients that are very early on in the course of pathology (NIS-LL < 10), although with this small cohort, and a correlation with the NIS-LL was not observed. In one embodiment, the ability of probe B-2 to detect circulating fragmented TTR in a subset of polyneuropathy patients allows a skilled artisan in the art to understand whether particular variants are more susceptible to proteolysis, which in turn could change the type of TTR aggregates that are formed, leading to unique proteotoxicity mechanisms and potentially explaining why there is early onset vs. late onset V30M FAP disease or why men typically progress faster than women in FAP.

[0098] In one embodiment, probe B-2 also provides the data for understanding the tissue tropism of the TTR amyloid diseases. A one amino acid change in the TTR sequence determines whether non-native TTR oligomers are detected in patient plasma, suggesting that distinct aggregate structures influence whether the peripheral nervous system or the heart is compromised by a given TTR sequence. Probe B-2 is not able to detect circulating non-native TTR oligomers in the plasma of patients harboring cardiomyopathy associated mutations, suggesting that a unique aggregate structure and rapid heart deposition may contribute to the distinct disease etiology in cardiomyopathy. Evidence from other amyloid diseases indicates that conformational differences within amyloid fibrils themselves may be linked with different disease phenotypes. Peptide-based probes for soluble Αβ oligomers have been developed and exhibit oligomer selectivity.

[0099] In one embodiment, the selectivity of the covalent peptide probe B-2 for non- native oligomeric TTR may be useful as an early diagnostic strategy for FAP or could be used as a response-to-therapy biomarker in polyneuropathy, as indicated within for tafamidis treatment / liver transplant-mediated gene therapy, that both slow the progression of FAP. As would be readily appreciated and understood by a skilled artisan, the probe B-l fluorescence- based assay or the probe B-2 photo-crosslinking approach can be elaborated beyond the methods disclosed herein, and such methods are contemplated by the present disclosure. As one non-limiting example, it is contemplated that the probes disclosed herein may be useful in an ELISA format, commonly used by clinical laboratories, rendering these peptide probes generally useful.

[00100] In some embodiments, the probe peptides also encompass variants, analogs, peptidomimetics or other derivative compounds that can be generated from the specific peptide sequences exemplified herein. These derivative compounds can be subject to the assays described herein to ascertain their binding activity for misfolded TTR oligomer. As noted above, the probe peptides typically contain from 6 amino acid residues to about 20 amino acid residues. Thus, in addition to the specific peptide sequences or motifs described herein, peptides that are suitable for probing misfolded TTR oligomer can also include one or more additional residues at the N-terminus and/or the C-terminus. In some embodiments, the derivative peptides are modified versions of the exemplified peptides which are generated by conservative amino acid substitutions. In some other embodiments, the derivative peptides are variants produced by non-conservative substitutions to the extent that that they substantially retain the binding activity of the exemplified peptides. In some embodiments, the analogs or derivative peptides of an exemplified probe peptide (e.g., SEQ ID NOs: 2, 10, 11 or 21) can contain one or more naturally occurring amino acid analogs or derivatives of the twenty standard amino acids, for example, 4-hydroxyproline, 5-hydroxylysine, 3- methylhistidine, homoserine, ornithine or carboxyglutamate. They may also include amino acids that are not linked by polypeptide bonds. Similarly, they can also be cyclic polypeptides and other conformationally constrained structures. Methods for modifying a polypeptide to generate analogs and derivatives are well known in the art, e.g., Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341, Academic Press, Inc., New York, N.Y. (1983); and Burger 's Medicinal Chemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620, John Wiley & Sons Inc., New York, N.Y. (1995).

[00101] In some embodiments, the disclosure provides probe peptidomimetics that are derived from the exemplified peptide sequences. Peptidomimetics based on an exemplified probe peptide (e.g., SEQ ID NOs: 2, 10, 11, or 21) substantially retain the activities of the reference peptide. They include chemically modified peptides, peptide-like molecules containing non-naturally occurring amino acids, peptoids and the like, have a structure substantially the same as the reference peptide upon which the peptidomimetic is derived (see, for example, Burger's Medicinal Chemistry and Drug Discovery, 1995, supra). For example, the peptidomimetics can have one or more residues chemically derivatized by reaction of a functional side group. In addition to side group derivatizations, a chemical derivative can have one or more backbone modifications including alpha-amino substitutions such as N-methyl, N-ethyl, N-propyl and the like, and alpha-carbonyl substitutions such as thioester, thioamide, guanidino and the like. Typically, a peptidomimetic shows a considerable degree of structural identity when compared to the reference peptide or polypeptide, and exhibits characteristics which are recognizable or known as being derived from or related to the reference polypeptide. Peptidomimetics include, for example, organic structures which exhibit similar properties such as charge and charge spacing characteristics of the reference polypeptide. Peptidomimetics also can include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid functional groups. [00102] In some other embodiments, the peptides described herein can include modifications within the sequence, such as, modification by terminal -NH ₂ acylation, e.g., acetylation, or thiogly colic acid amidation, by terminal-carboxylamidation, e.g., with ammonia, methylamine, and the like terminal modifications. Terminal modifications are useful to reduce susceptibility by proteinase digestion, and therefore can serve to prolong half-life of the polypeptides in solution, particularly in biological fluids where proteases may be present. Amino terminus modifications include methylation (e.g., -NHCH ₃ or -N(CH ₃ ) ₂ ), acetylation (e.g., with acetic acid or a halogenated derivative thereof such as a-chloroacetic acid, a-bromoacetic acid, or a-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO- or sulfonyl functionality defined by R-SO2-, where R is selected from the group consisting of alkyl, aryl, heteroaryl, alkyl aryl, and the like, and similar groups. One can also incorporate a desamino acid at the N-terminus (so that there is no N-terminal amino group) to decrease susceptibility to proteases or to restrict the conformation of the peptide compound. In some embodiments, the N-terminus is acetylated with acetic acid or acetic anhydride. Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints. One can also incorporate a desamino or descarboxy residue at the termini of the peptide, so that there is no terminal carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the peptide. Other functional groups to modify the C-terminus include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.

[00103] The probe peptides described herein, including variants and derivatives thereof, can be chemically synthesized and purified by standard chemical or biochemical methods that are well known in the art. Some of the methods are described in the Examples herein. Thus, in some embodiments, solid phase peptide synthesis can be employed for producing the probe peptides and their derivative compounds. In some embodiments, the peptides may be synthesized using t-Boc (tert-butyloxycarbonyl) or FMOC (9-flourenylmethloxycarbonyl) protection group described in the art. See, e.g., "Peptide synthesis and applications" in Methods in molecular biology Vol. 298, Ed. by John Howl; "Chemistry of Peptide Synthesis" by N. Leo Benoiton, 2005, CRC Press, (ISBN-13: 978-1574444544); and "Chemical Approaches to the Synthesis of Peptides and Proteins" by P. Lloyd-Williams, et. al, 1997, CRC-Press, (ISBN-13: 978-0849391422), 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. In some embodiments, the probe peptides and derivatives thereof may also be synthesized and purified by recombinant methods that are well known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3 ^rd ed., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003).

IV. Diagnostic moieties for labeling TTR-derived peptide probes

[00104] To facilitate detection of misfolded TTR oligomers upon their binding to the probe peptide, the probe compound typically contains one or more detectable labels (label moieties or diagnostic moieties). A detectable label is a molecule or functional group that can itself be detected or can produce a detectable signal upon reacting with another molecule under appropriate conditions. It can be detected by a variety of methods including fluorescence, electrical conductivity, radioactivity, size, and the like. The label may be directly or indirectly detectable. For example, the label can be detected directly by its ability to emit and/or absorb light of a particular wavelength. A label can be detected indirectly by its ability to bind, recruit and, in some cases, cleave (or be cleaved by) another compound, thereby emitting or absorbing energy. One example of indirect detection is the use of a reporter function group (e.g., an alkyne group) which can react with and become attached to a reporter molecule (e.g., rhodamine-azide). Another example of indirect detection is the use of an enzyme substrate that can be cleaved to produce detectable products. The type of label moieties or diagnostic moieties that may be used are typically sterically and chemically compatible with the peptide component of the probe compound. In general, the label should not interfere with the folding or other activity of the target TTR oligomer protein.

[00105] For labeling the peptide probe, the detectable label does not include a peptide sequence that is present in the natural TTR protein. In various embodiments, the label in the probe compounds can be a fluorescent molecule, a chemiluminescent molecule (e.g., chemiluminescent substrates), a phosphorescent molecule, a radioisotope, an enzyme substrate, an affinity molecule, a ligand, an antigen, a hapten, an antibody, an antibody fragment, a chromogenic substrate, a contrast agent, an MRI contrast agent, a positron emission tomography (PET) label (e.g., Technetium-99m and fludeoxy glucose), a phosphorescent label, and the like. However, to ensure binding of the probe compound to the TTR oligomer protein, the detectable label is preferably a small moiety such as a detectable atom (e.g., a radioactive isotope), a small organic molecule, or a small reactive chemical moiety or functional group, as opposed to bigger molecules such as enzymes or other polypeptides. Thus, in some preferred embodiments, the detectable label in the probe compounds can be a fluorescent group or moiety as exemplified herein. In some preferred embodiments, the detectable label can be a photoactivatable cross-linker. In some other embodiments, the label moiety in the probe compounds can be radioactive isotopes such as 2P or H. In some embodiments, the detectable label in the probe compounds can be haptens such as digoxigenin and dintrophenyl. The detectable label can also be an analyte-binding group such as but not limited to a metal chelator (e.g., a copper chelator). The label may also be a heavy atom carrier such as iodine, Au, Pt and Hg.

[00106] In some preferred embodiments, the detectable labels or label moieties in the probe compounds are fluorescent, luminescent, or absorbent label moieties. These label moieties include fluorophores, rhodamine moieties, and coumarin moieties (e.g., such as 7- amino-4-carbamoylcoumarin, 7-amino-3-carbamoylmethyl-4-methylcoumarin, or 7-amino-4- methylcoumarin). Examples of fluorophores that can be used include, e.g., fluorescein, fluorescein analogs, BODIPY-fluorescein, arginine, rhodamine-B, rhodamine-A, rhodamine derivatives, and the like. For further information on fluorescent label moieties and fluorescence techniques, see, e.g., Handbook of Fluorescent Probes and Research Chemicals, by Richard P. Haugland, Sixth Edition, Molecular Probes, (1996). In some embodiments, the detectable label is a long wavelength fluorophore such as fluorescent dyes in the Alexa Fluor family (Thermo Fisher Scientific Inc.). In some exemplary embodiments as described herein, a fluorescein derivative such as 5-FAM-X-SE (6 - (Fluorescein - 5 - carboxamido)hexanoic acid, succinimidyl ester) is used to label the probe compounds. This will result in the probe peptide being conjugated to a fluorophore, 6-(fluorescein-5-carboxamido)hexanoic acid (5- FAM-X). An example of such peptide probes is Probe Bl as exemplified herein. Probe Bl contains Peptide AINVAVHVFR (SEQ ID NO: 2) conjugated to a fluorophore diagnostic moiety 5-FAM-X (X refers to the hexanoic acid linker).

[00107] In general, the detectable label can be attached to the probe peptide at any position. In some embodiments, the detectable label is attached to the N-terminal residue of the probe peptide. The attachment can be either covalent or non-covalent. As exemplified herein, some probe compounds have a detectable label that is covalently bonded to the N- terminus of the probe peptide. Preparation of fluorophore-labeled peptide probes can be readily performed via protocols exemplified herein and/or method well known in the art. See, e.g., Weder et al., J. Chromatogr. 698, 181, 1995; Cavrois et al, Nat. Biotechnol. 20, 1151-1154, 2002; and Marme et al, Angew. Chem., Int. Ed. Engl. 43, 3798, 2004. In general, labeled peptide probes can be prepared by either modifying isolated peptides or by incorporating the label during solid-phase synthesis. For example, fluorophores can be conjugated to the N-terminus of a resin-bound peptide before other protecting groups are removed and the labeled peptide is released from the resin. Labeling of the peptide probes can also be achieved indirectly by using a biotinylated amino acid. For example, when Fmoc-Lys(biotinyl)-OH is used in peptide synthesis, the biotin group allows specific binding of streptavidin or avidin-conjugate to that site. A variety of fluorophores are available as (strept)avidin conjugates. In some other embodiments, quantum dots can be employed as label moieties. For example, semiconductor nanocrystals or quantum dots such as cadmium selenide and cadmium sulfide can be used as fluorescent probes. See, e.g., Bruchez et al, Science 281 :2013-2016, (1998). In some other embodiments, the label moieties in the probe compounds are electroactive species for electrochemical detection or chemiluminescent moieties for chemiluminescent detection. UV absorption is also an optional detection method, for which UV absorbers are optionally used. Phosphorescent, colorimetric, e.g., dyes, and radioactive labels can also be optionally attached to the probe peptides.

[00108] In some other preferred embodiments, the probe compounds can contain a diagnostic moiety or label moiety that is a photoactivatable cross-linker. A photoactivable cross linker is a photo-affinity group that becomes reactive upon exposure to radiation (e.g., a ultraviolet radiation, visible light, etc.). Examples include diazirine functional groups, benzophenones, aziridines, a photoprobe analog of geranylgeranyl diphosphate (2-diazo- 3,3,3-trifluoropropionyloxy-farnesyl diphosphate or DATFP-FPP) (Quellhorst et al. J Biol. Chem. 2001 Nov. 2; 276(44):40727-33), a DNA analogue 5-[N-(p-azidobenzoyl)-3- aminoallyl]-dUTP(N(3)RdUTP), sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3- acetamido)-ethyl-l,3'-dith-iopropionate (SAED) and l-[N-(2-hydroxy-5-azidobenzoyl)-2- aminoethyl]-4-(N-hydroxysuccinimidyl)-succinate. As exemplified herein, non-native misfolded TTR oligomers can be readily detected via photo-affinity labeling of the probe compounds with such photo-reactive functional groups. Photo-affinity labeling (PAL) has been well known for target identification in complex protein mixtures. Typically, PAL utilizes a biologically active small-molecular photo-affinity probe that bears photo-reactive and reporter functional groups to identify macromolecular binding partners. The photo- affinity probe is designed and synthesized based on SAR (structure-activity relationships) of a parent small-molecule having known biological activity. During PAL, the photo-affinity probe is incubated with a protein mixture and irradiated with UV light. Irradiation of the photo-reactive group generates a highly reactive chemical species (e.g. carbene, nitrene, or radical) that covalently crosslinks the photo-affinity probe to its macromolecular binding partners. Photo-crosslinked protein targets are then visualized by the reporter group (e.g. fluorophore, biotin, or radioactive label). Covalent bond formation between the probe and targets enable the subsequent purification and identification of the targets using techniques such as SDS-PAGE, immunoprecipitation, biotin-streptavidin affinity purification and mass spectrometry.

[00109] In some embodiments, the probe peptide is labeled with an alkyl diazirine photo- reactive group. An example of such probes, Probe B2, diazirine - propargyl glycine (alkyne) -INVAVHVFR (SEQ ID NO:22), is exemplified herein. It was generated by conjugating the N-terminal residue of peptide INVAVHVFR (SEQ ID NO: 11) with two label moieties, an alkyne reporter group followed by an alkyl diazirine reactive group. Alkyl diazirine is compact in size, being nearly isosteric to a methyl group, and is accessed synthetically via an alkyl ketone. This allows installation of the diazirine at positions of a small-molecule that would not tolerate larger, aryl-based photo-reactive groups. The carbene intermediate formed upon photo-activation of the diazirine can rapidly insert into X-H bonds (X = N, S, O), as well as C-H bonds, to form stable covalent insertion products. When not poised for insertion into bonds of the macromolecular target, the alkyl carbene intermediate undergoes rapid quenching by solvent or internal rearrangement to a stable olefin side-product. The alkyl diazirine is also stable toward acidic and basic conditions and toward ambient light encountered during routine chemical synthesis. In the practice of the present disclosure, heterobifunctional amine-reactive alkyl diazirine crosslinkers, as well as alkyl diazirine- containing amino acid analogs can be obtained commercially, e.g., from Pierce or Thermo Scientific. Labeling a peptide with a diazirine functional group can be readily performed in accordance with the protocols described herein or other methods routinely practiced in the art. See, e.g., MacKinnon et al, Curr. Protoc. Chem. Biol. 1 : 55-73, 2009; Bond et al., Nat. Protoc. 4: 1044-63, 2009; and MacKinnon et al., J. Am. Chem. Soc. 129: 14560-14561, 2007.

[00110] In addition to the photoreactive functional group, the probe compound can additionally include a diagnostic moiety that is a reporter function group for conjugation to a detectable reporter compound, e.g., an alkyne reporter group as exemplified herein for Probe B2. In these embodiments, detection of a target protein with a photo-affinity labeled probe compound can be carried out via, e.g., Cu(I)-catalyzed click chemistry (see, e.g., Wang et al. J. Am. Chem. Soc. 125: 11164-11165, 2003). This method allows bio-conjugation of probe- labeled proteins with reporter groups. During the click reaction, Cu(I) catalyzes a highly selective, bio-orthogonal 1,3 dipolar cycloaddition reaction between a terminal alkyne group and an azide group. This results in the formation of a stable triazole product. As exemplified herein, the terminal alkyne is typically present in the labeled probe compound, while the azide is present in a fluorescent or biotinylated reporter compound, e.g., Rhodamine-azide or biotin-azide. Alternatively, the azide can be incorporated into the probe and the alkyne incorporated into the reporter. As demonstrated in the Examples herein, the diazirine and azide labeled probe compound covalently labels the TTR oligomer protein upon irradiation, which is then further conjugated to the azi de-bearing reporter under click chemistry conditions. In addition to the alkyne group for labeling the probe compound that Rhodamine- azide reporter exemplified herein, many other azide function group and alkyne-reporters that are known in the art for bio-conjugate click reactions may also be utilized. See, e.g., Speers et al, Chem Biol. 11 :535-46, 2004.

[00111] In some other embodiments, the probe compounds can contain a photolabile protecting group or a photoswitch label. Photolabile protecting groups are useful for photocaging reactive functional groups. Examples of photolabile protecting group include a nitrobenzyl group, a dimethoxy nitrobenzyl group, nitroveratryloxycarbonyl (NVOC), 2- (dimethylamino)-5-nitrophenyl (DANP), Bis(o-nitrophenyl)ethanediol, brominated hydroxy quinoline, and coumarin-4-ylmethyl derivative. A photoswitch label is a molecule that undergoes a conformational change in response to radiation. For example, the molecule may change its conformation from cis to trans and back again in response to radiation. The wavelength required to induce the conformational switch will depend upon the particular photoswitch label. Examples of photoswitch labels include azobenzene, 3-nitro-2- naphthalenemethanol. Examples of photoswitches are also described in van Delden et al. Chemistry. 2004 January 5; 10(l):61-70; van Delden et al. Chemistry. 2003 June 16; 9(12):2845-53; Zhang et al. Bioconjug Chem. 2003 July-August; 14(4):824-9; Irie et al. Nature. 2002 December 19-26; 420(6917):759-60; as well as many others.

[00112] Depending on the specific probe compounds being used, binding of the probe compounds to target misfolded TTR oligomer proteins can be either non-covalent or covalent. The binding can be detected via various detection systems. Detection systems may be selected from a number of detection systems known in the art. These include a fluorescent detection system, a photographic film detection system, a chemiluminescent detection system, an enzyme detection system, an atomic force microscopy (AFM) detection system, a scanning tunneling microscopy (STM) detection system, an optical detection system, a nuclear magnetic resonance (NMR) detection system, a near field detection system, and a total internal reflection (TIR) detection system. The specific detection system to be used will depend upon the nature of the detectable labels in the probe compounds. For example, binding to a target protein by a probe compound containing a photo-affinity label and a reporter function group (e.g., rhodamine- or biotin-conjugated azide) can be first subject to photo-crosslinking. This is followed by performing appropriate assays to isolate (e.g., via size exclusion chromatography) and detect the protein complexes (e.g., via electrophoresis and fluorescence imaging, or Streptavi din-based precipitation and Western blot).

[00113] In some embodiments, the detection system can be based on an ELISA-like assay. In this detection system, the biological sample can be first contacted with a probe compound that can pull down the misfolded TTR oligomers. The isolated probe-TTR complexes can then be analyzed and quantified in an ELISA-like plate format. In addition to detecting misfolded TTR oligomers in biological sample, this detection system can be used to examine any other molecules that specifically interact with the non-native TTR oligomer protein. As exemplified herein, one such TTR interacting protein partner is the extracellular chaperone Clusterin. Some of these embodiments can employ probe B-2 exemplified herein which is labeled with a photoreactive group and a terminal alkyne reporter group. Following irradiation to crosslink the probe to the TTR oligomer and reacting with a biotin-azide reporter molecule via click chemistry, the complexes can be affinity purified via streptavidin- biotin interaction. The isolated non-native TTR oligomers from the biological sample can then be further analyzed on a plate with antibodies to TTR, Clusterin and other related molecules. With this assay system, multiple biological samples can be analyzed simultaneously for the presence of different target molecules of interest. By analyzing the presence of the different target molecules, this assay format also enables the generation of a risk score for the subject whose biological sample was tested.

[00114] In some other embodiments, binding of fluorescein-labeled probes to the misfolded TTR oligomer can be detected via monitoring fluorescein fluorescence following sample separation by gel electrophoresis or gel filtration chromatography analysis. In other embodiments, binding of the probe compounds to the target TTR protein may be determined by a number of other methods, e.g., quenching and other intensity measurements, donor or acceptor depletion kinetics, and fluorescence lifetime or emission anisotropy measurements.

V. Detecting non-native TTR oligomers and diagnosis of TTR amyloid diseases

[00115] The present disclosure provides methods for detecting misfolded TTR oligomeric proteins, related applications for diagnosing TTR amyloid diseases, as well as methods for monitoring disease status or therapeutic effects in patients with TTR amyloid diseases. As used herein, TTR amyloid diseases encompass transthyretin-related hereditary amyloidoses (ATTR). Depending on the sequence of TTR that misassembles, these hereditary disorders include conditions with predominant degeneration of the heart (cardiomyopathy), the peripheral nervous system (polyneuropathy), the central nervous system (meningocerebrovascular amyloidosis), and the eye (ocular amyloidosis). Two specific examples of these autosomal dominant neurodegenerative diseases are familial amyloid polyneuropathy (FAP) and familiar amyloid cardiomyopathy (FAC). Unless otherwise noted, TTR amyloid diseases suitable for the diagnostic methods disclosed herein 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.

[00116] 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 amyloidoses, with the most common mutation worldwide being TTR Val30Met (V30M TTR) and TTR Vall22Ile (VI 221 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.

[00117] 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 Vall22Ile (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.

[00118] 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. 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 carpal tunnel 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.

[00119] The diagnostic methods disclosed herein 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 probe compounds can detect non-native TTR oligomers in patients with TTR aggregation associated amyloidoses - both polyneuropathy and cardiomyopathy phenotypes. An important aspect of the probe compounds is that, unlike other probes used for diagnostic purposes (i.e. Congo Red or thioflavin T), they preferentially recognize nascently unfolded and actively aggregating TTR. As a result, the diagnostic methods described herein are suitable for detection of the earliest possible misfolding events that lead to TTR related amyloid diseases.

[00120] To detect and/or quantify misfolded (i.e., non-native) TTR oligomer, a suspected biological sample (e.g., a blood sample) is typically contacted and incubated with a probe compound. Upon incorporation of the probe peptide into the misfolded TTR oligomer, the complex can be readily detected and examined via routinely practiced assays. As exemplified herein, the exact assay to be employed in the detection is dependent on the nature of the diagnostic moiety present in the probe compound. Suitable biological samples include, but are not limited to, blood samples, cerebrospinal fluid, vitreous fluid, tears, ascites, sweat, urine, saliva, buccal sample, cavity or organ rinse. In some preferred embodiments, detection of misfolded TTR oligomer in candidate subjects having or suspected of having a TTR amyloidosis is performed with a blood sample from the candidate subjects. As exemplified herein, blood samples including whole blood, blood plasma or blood serum can be readily employed for detection of misfolded TTR oligomer. If misfolded TTR oligomer is positively detected in the biological sample relative to a control as described below, the candidate subject is identified as one who likely has or is likely to develop a TTR amyloidosis disease or condition. [00121] In some embodiments, the diagnostic methods disclosed herein can be employed in conjunction with other diagnostic methods known in the art. The diagnostic test disclosed herein 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 disclosed herein. Upon a positive test with the diagnostic method disclosed herein, the patients with positive test results can then 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.

[00122] In addition to diagnosis applications for screening or early detection of misfolded TTR oligomers, the probe compounds can also be employed in assessing disease status or progression, and monitoring effect of treatments that TTR amyloidosis patients are undergoing. Subjects suitable for these methods 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 for these treatments. For example, medications that may help to 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 probe compounds can be employed to assess disease status or monitor treatment effect in patients undergoing any of these treatments.

[00123] Depending on the diagnostic moiety or detectable label that is present on the probe compound, various assays can be employed in the methods disclosed herein. Typically, detection and quantification of misfolded TTR oligomer via a chosen assay is based on a detected signal level indicative of the presence of non-native TTR oligomer relative to a background or control signal level determined via the same assay. In some embodiments, the background or control signal level is determined with the same type of biological sample that is known not to contain misfolded TTR oligomer (e.g., biological sample from healthy subject). In some embodiments, the control or background may be determined via a control probe peptide that does not bind to misfolded TTR oligomer. If the detected signal level does not differ significantly from the control signal level, the outcome of the diagnostic assay is considered negative. On the other hand, if there is a significant departure between the detected signal level in a biological sample from a candidate subject and the control signal level, it indicates a positive outcome of the diagnostic test. Additionally or alternatively, the assays can further include comparing the detected signal level in the candidate subject with a positive standard or control signal level. 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 detected signal level and the positive control signal level. For example, a positive diagnosis can be established by a detected signal level that is comparable to or falls within the range of positive control signal levels determined from a population of subjects affected by a TTR amyloidosis.

[00124] A departure is considered significant or substantial if the detected signal level 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 detected signal level does not fall within the mean plus one standard deviation of levels in a control population. Typically, a significant departure occurs if the difference between the detected signal level and background or control levels is at least 20%, 30%, or 40%. Preferably, the difference is by at least 50% or 60%. More preferably, the difference is more than at least 70% or 80%. Most preferably, the difference is by at least 90%. The extent of departure between a detected signal value and a background or control value 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.

[00125] In some embodiments for monitoring disease progression or treatment effect, signal levels via the same assay are detected 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 to monitor 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 these embodiments, an increase of over 10%, 20%, 30%, 40%, 50%, 100%, 200%, 500% or more of a detected signal level 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. A decrease of a detected signal level (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more) at a later time point relative to a signal level 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.

EXAMPLES

[00126] 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

Methods and materials

[00127] This Example describes some materials and methods employed in the exemplified embodiments herein.

[00128] Study Design: The objective of the instant study was to develop peptide-based probes for transthyretin oligomers and to show proof-of-principle of their utility in detection of oligomers in the plasma of patients with hereditary amyloidosis. All patient samples and analyses were collected under the approved IRB protocols at their respective institutions and no blinding or randomization was used. The number of unique clinical plasma samples for each experiment is indicated in the respective figures and legends. Plasma from healthy blood donors and asymptomatic mutation carriers were chosen as controls. Sample sizes in the range of 10-50 were chosen for feasibility in accrual and provided a reasonable level of statistical power for comparison of the three groups.

[00129] Peptide synthesis and purification: Peptides were synthesized by solid-phase peptide synthesis using a standard Fmoc a-amine protecting group strategy (Applied Biosystems Model 433A). Resin and amino acids used for peptide synthesis were purchased from Novabiochem Corp (San Diego, CA, USA). HOBt (1-hydroxy-benzotriazole; Advanced Chem Tech), HBTU (N,N,N',N'-Tetramethyl-0-(lH-benzotriazol-l-yl)uronium hexafiuorophosphate, 0-(Benzotriazol- 1 -yl)-N,N,N'N'-tetramethyluronium hexafluorophosphate; Sigma Aldrich), and DIEA (Ν,Ν-diisopropylethylamine; Sigma Aldrich) were used as coupling reagents, and piperidine (Sigma Aldrich) was used for the standard deprotection method.

[00130] The peptides were cleaved from the resin using Reagent K (87.5 : 5 : 5 : 2.5; trifiuoroacetic acid, thianisole, water, 1,2-ethanedithiol). Peptides were synthesized on a Applied Biosy stems 433 A automated peptide synthesizer using a standard FMOC protection strategy. Typically after synthesis, the cleaved peptide was precipitated using cold diethylether, recovered by centrifugation, reconstituted in anhydrous DMF or DMSO and incubated with 5-FAM-X-SE (6 - (Fluorescein - 5 - carboxamido)hexanoic acid, succinimidyl ester) (Anaspec Inc.) or SE-Diazirine (succinimidyl 4,4'-azipentanoate) for two hours in the presence of DIEA (1 : 100 DIEA/DMF). For sequences that contain lysine, fluorophore coupling was done on the resin prior to deprotection and cleavage. Purification of labeled peptides was achieved using preparative C18 RP-HPLC under acidic conditions (Buffer A: 0.1% TFA in water, Buffer B: 0.1% TFA in acetonitrile). Peptides masses were confirmed by LC-MS. After purification the fractions containing the peptide of interest were lyophilized and stored under dessicant at -20 °C. Prior to each experiment peptides were freshly reconstituted in DMSO for use.

[00131] Expression and purification of recombinant TTR: Recombinant wild-type and mutant TTR (M-TTR corresponding to F87M/L110M TTR mutant, A25T, V30M, V122I, L55P, T119M) were expressed and purified from E. coli. The C-terminal TTR fragments TTR5 ₀ -127 and/or TTR52-127 were expressed and purified as follows. The protein coding sequence was cloned into a plasmid upstream of an IPTG inducible T7 promoter (pMMHa). E. coli BL21 containing the TTR5 ₀ -127 and/or TTR52-127 plasmid were grown to an OD of 0.6 and induced at 37 °C with 1 mM IPTG and the protein was allowed to express overnight. After induction and protein expression the majority of the protein was found within inclusion bodies. The E. coli cells were lysed by sonication and centrifuged at 15,000 x g. The supernatant was discarded and the insoluble fraction was resuspended in 10 mL 6M guanididium-HCl and sonicated for 35 minutes in a water bath, followed by vigorous stirring for 2 hrs. The mixture was then centrifuged again at 15,000 xg for 15 minutes. The supernatant was then taken and dialyzed against 4L of 25 mM Tris pH 8.8, followed by injection onto a Sourcel5Q strong anion exchange column (GE Healthcare ). A linear gradient was then run to elute the protein (Buffer A 25 mM Tris, 2M Urea pH 8.8, Buffer B: 25mM Tris pH 8.8, 2M Urea, 1M NaCl). The fractions containing TTR50-127 and/or TTR52-127 were then concentrated and injected onto a sephadex 75 gel filtration column, run in 25 mM Tris pH 8.8, 2M Urea. The purified protein was then dialyzed into 5 mM NH4HCO4, frozen on liquid N ₂ and lyophilized.

[00132] Production of non-native recombinant TTR oligomers and aggregates: A25T-TTR and MTTR readily form stable oligomers at neutral pH. Briefly, the proteins were concentrated to 2 mg/mL in lOmM sodium phosphate pH 7.6, lOOmM KC1, lmM EDTA and incubated at 37 °C for 7 days ("young") or 6 weeks ("old"). To make aggregates of TTR52-127, 0.5 mg of the protein powder was dissolved in 10 mM sodium phosphate pH 7.6, 100 mM KC1, 1 mM EDTA to a final concentration of 70 μΜ. the solution was placed in sonicating water bath for 5 minutes to fully dissolve the peptide an then passed through a 0.2 μιτι filter and incubated at room temperature for 4 hours. Soluble aggregates were found to form within hours (Fig. 9).

[00133] For the peptide screen, to quantify peptide incorporation into recombinant oligomers (TTR5 ₀ -127 and MTTR), each candidate probe (2 μΜ) was incubated overnight with 50 μΜ oligomeric TTR followed by injection onto an Agilent SEC-3 or SEC-5 column and monitored for fluorescence (495 ex., 520 em.). A fluorescein standard curve was used to compare results from day to day. The results represent the area of the fluorescently labeled high MW peak.

[00134] To determine the stoichiometry, 20 μΜ of the B-1 peptide (the identical concentration of probe used in analysis of the patient samples) was incubated with 50 μΜ MTTR oligomerized for different time periods, and then the high MW labeled peak was separated using the same SEC method as above. The high MW fraction was denatured using guanidinium chloride and the amount of B-1 was measured by the fluorescence intensity of the denatured sample on an Aviv ATF 105 spectrofluorimeter at 25 °C, using an excitation wavelength of 490 nm and recording emission spectra between 600 and 500 nm. A standard curve with B-1 in the same denaturing conditions was used to determine the amount of B-1 in the high MW fraction, which was then divided by the amount of protein in that fraction. To determine quenching, an emission spectrum was taken prior to denaturation and compared to a B-1 emission spectrum at the determined concentration in the denaturation experiment. This procedure is depicted in fig. 23. [00135] Collection of Plasma samples: Blood from healthy volunteers was obtained from The Scripps Research Institute's Normal Blood Donor Services Center. The blood was collected in BD Vacutainer tubes prepared with heparin and allowed to sit upright for at least 30 min at room temperature. The blood was then centrifuged at 1500g for 20 min. The resulting supernatant (plasma) was carefully removed and centrifuged for an additional 20 min to remove any remaining cells. Aliquots of the clarified plasma were stored at -20 °C. Blood from V30M FAP patients (from Portugal and Japan), asymptomatic V30M mutation carriers (from Portugal) and Portuguese and Japanese controls were also collected in anticoagulant coated tubes, and plasma was prepared in the same way at the corresponding center. Remaining blood samples (from WT cardiomyopathy patients and the non-V30M patients) were collected at Columbia University College of Physicians and Surgeons (NY) and at the Boston Amyloidosis Center. Plasma was transferred to 1.5 mL cryovials with a cap and stored at -75 °C until it was shipped. After shipment in dry ice, frozen patient plasma samples were kept at -20°C. Frozen plasma samples were thawed on ice before being used. This study was approved by the respective Institutional Review Boards in Portugal, Japan, NY, Boston and The Scripps Research Institute, and informed consent was obtained from the participants.

[00136] Analysis of non-native recombinant oligomers / recombinant tetramers by NATIVE gel: Formation of oligomers was monitored using Native-gels 4-16% (Novex): 20- 30uL of each sample was loaded into the gel, ran for 100 min at 150V in native conditions, and stained with Comassie Blue or silver stain. In the experiments were different peptides were incubated with a recombinant non-native TTR oligomers, the gels were scanned on a Bio-Rad ChemiDoc system using the predefined fluorescein channel, before staining for total protein.

[00137] Identification of plasma high molecular weight structures in patient plasma: Plasma from i) symptomatic V30M neuropathy patients, ii) symptomatic wt-ATTR cardiomyopathy patients, iii) healthy controls, and iv) asymptomatic V30M carriers (45μί) was incubated with B-1 (20μΜ; 3.2μΙ, of B-1 from a 300μΜ stock solution in DMSO) or 3.2μΙ _^ of DMSO for 24 hours at 37°C. Before injecting onto the size exclusion column (BEH200, Waters™), each plasma sample was passed through a P30 spin gel filtration column (BioRad) to remove excess unbound peptide and preserve the lifespan of the column (final volume = 50 μί). 5 μΐ. of each sample was then injected onto the column and separated using a constant flow of 0.2mL/min of lOmM phosphate buffer, lmM EDTA, pH 7.8, for 30 minutes. To account for plasma auto-fluorescence, the resulting signal from the samples with no B-l peptide (DMSO only) was subtracted from the signal from the corresponding sample with the B-l peptide. At the start and the end of each sample set, four injections of different concentrations of Fluorescein in the running buffer (freshly prepared each day) were analyzed by the same instrument, in order to make a standard curve for day-to-day comparison. The amount of B-l in the high MW weight fraction was then calculated from the slope of the standard curve ([Fluorescein] vs. Fluorescence (ex495, em520)). Finally, all data were then normalized to the total protein concentration as calculated from the integrated area in the 280 nm absorbance channel.

[00138] Diazirine photo-crosslinking and pulldown: B-2 was synthesized and purified as mentioned above. Lyophillized B-2 powder was weighed and dissolved in anhydrous DMSO to a final stock concentration of 1 mM. This stock was aliquoted and stored at -80 °C. For each experiment a fresh aliquot of B-2 was taken and added directly to the sample of interest (plasma, buffer etc.) to a final concentration of 50 μΜ and incubated at 37 °C overnight. Time course experiments with plasma from patients and MTTR within plasma from healthy donors, showed that the signal plateaued within 5-8 hours and did not change thereafter. After incubation, plasma was pipetted into a 96 well plate and irradiated at 355 nm in a Stratalinker 1800 at 4°C for 1 hr with the lid of the plate off. After photoactivation and crosslinking, free peptide was removed and buffer exchanged into 10 mM Sodium Phosphate buffer (pH 7.6), 100 mM KCl using a spin gel filtration column (P30 resin, Biorad). 10 uL of the sample was then injected onto a BEH200 (waters) or a SEC3 (Agilent, higher loading capacity) size exclusion column (mobile phase 10 mM Sodium Phosphate buffer (pH 7.6), 100 mM KCl). Both columns used separate biomolecules in the range of 100-600 kDa while the Agilent SEC3 has a higher loading capacity. Aliquots of the samples either before or after SEC were conjugated with either Rhodamine-azide or Diazo-Biotin-azide.

[00139] For rhodamine conjugated samples. Each sample was first MeOH/CHC13 precipitated and resuspended in 1 x Laemilli buffer containing either TCEP or DTT. After resuspension the samples were boiled for 5 min and loaded onto a 4-12% precast denaturing gel (4-12% Bolt™, Life Technologies) and run at 200V for 30 min. The gel was then imaged using the rhodamine filter on a Biorad XR+ gel imaging system.

[00140] For biotin conjugated samples. After the CuACC reaction, each sample was MeOH/CHC13 and resuspended in 500 uL of 8M Urea, IX PBS, 5 mM EDTA, followed by the addition of 140 uL of 10% SDS. Following resuspension the samples were then reduced by adding TCEP (10 mM final concentration) and incubated for 30 min at room temperature. The free sulfhydryls were then blocked by adding lodoacetamide to a final concentration of 12 mM and incubated at room temperature for 30 minutes in the dark. After reduction and alkylation, the samples were then added to 6 mL of PBS containing 100 uL of Streptavidin conjugated agarose beads (Novagen) and incubated at room temperature with gentle agitation overnight. The biotin beads were then washed 6 x with 1 x PBS, 0.5% SDS, 5 mM EDTA and eluted with 100 uL of dithionite containing solution by incubation for 30 min at room temperature. The samples were then run in a denaturing gel for western blot analysis. For anti-TTR western analysis, the gel was transferred to nitrocellulose, blocked with Odyssey blocking buffer and probed with the anti-TTR antibody (rabbit, DAKO™). The membrane was then imaged using a Licor™ infrared imager by the addition of an anti-rabbit alexa680 conjugated secondary antibody.

[00141] Salivary gland biopsies staining with Thioflavin T. Anti-TTR antibody and B-l- Biotin: Deparaffinization and re-hydration was performed according to the following protocol: 2 x 5 min washes with 100% xylene, followed by 3 minutes wash with 50 : 50 xylene : ethanol, followed by 3 minutes washes with ethanol 100%, 95%, 70%, 50%, and 2 x 3 min with water. For Thioflavin T staining, slices were incubated for 10 minutes at room temperature with 1% aqueous Thioflavin-T (AnaSpec. Inc Ultra Pure Grade, Cat # 88306; filtered before each use with a 0.22 μιτι syringe filter). Following incubation with Thioflavin T, slices were washed 2 times with 80% ethanol, and then covered with coverslip in Vectashield® Mounting Medium. Slices were allowed to dry overnight in the dark. For anti- TTR antibody (rabbit anti-TTR, DAKOTM; pre-labeled with biotin) and B-l -Biotin staining, slices were submitted to a mild permeabilization protocol as follows: 2 x 5 min washes with cold methanol, followed by 2 x 5 min with PBS, 2 x 5 min with PBS-0.4% Triton® X-100 (Fisher Scientific), and 20 min with 0.05% saponin (Acros Organics, Cat# 41923-1000) and 3 x 5 min wash with PBS. Slices were then incubated overnight at room temperature with antibody (1 :200 dilution), B-l -Biotin probe (100 μΜ in DMSO) or DMSO (Vehicle). On the next day, slices were washed 3 x 10 min with PBS, followed by 1 hour incubation with Streptavidin Alexa®-594 Conjugated. Lastly, slices were washed 3 x 5 min with PBS, and covered with coverslip Vectashield® Mounting Medium. Slices were allowed to dry overnight in the dark. All slices were visualized using an Olympus 1X71 Inverted Microscope and representative photos were taken using an attached microscope camera Hamamatsu C8484-03G01 and the HCImage software. [00142] Incubation of plasma samples with probe B-l and size exclusion chromatography. Plasma (45 μί) was incubated with probe B-l (20 μΜ; 3.2 of probe B-l from a 300 μΜ stock solution in DMSO) or 3.2 μΐ. of DMSO for 24 hours at 37°C. Before injecting onto the size exclusion column (Acquity UPLC® Protein BEH SEC Column, 20θΑ, 1.7 μπι, 4.6 mm x 150 mm, Waters™), each plasma sample was filtered through a P30 gel filtration column (Bio-Spin® Columns with Bio-Gel® P-30) to remove excess unbound peptide and preserve the lifespan of the SEC column (final volume = 50 μί). 5 μΐ. of each sample was then injected onto the column and separated using a constant flow of 0.2 mL/min of 10 mM phosphate buffer, 1 mM EDTA, 100 mM KCl, pH 7.8, for 30 minutes. To account for plasma auto-fluorescence, the resulting signal from the samples with no probe B-l (DMSO only) was subtracted from the signal from the corresponding sample with the probe B-l . At the start and the end of each sample set, four injections of different concentrations of fluorescein in the running buffer (freshly prepared each day) were analyzed by the same instrument, in order to make a standard curve for day-to-day comparison. All data were then normalized to the total protein concentration as calculated from the integrated area in the 280 nm absorbance channel. The results of the different groups were compared using a two-tailed t- test.

[00143] Diazirine photocrosslinking and pulldown: Lyophilized probe B-2 powder was weighed and dissolved in DMSO to a final stock concentration of 1 mM and added to the plasma samples to give a final concentration of 50 μΜ and incubated at 37 °C overnight. Time course experiments with plasma from patients and MTTR oligomers within plasma from healthy donors, showed that the signal plateaued within 5-8 hours and did not change thereafter. After incubation, plasma was pipetted into a 96-well plate and irradiated at 355 nm in a Stratalinker® UV Crosslinker 1800 for 1 hour. After photoactivation and crosslinking, free peptide was removed and the probe B-2 conjugates buffer exchanged into 10 mM sodium phosphate buffer (pH 7.6), 100 mM KCl using a spin gel filtration column (Bio-Spin® Columns with Bio-Gel® P-30). 5-20 μΐ. of the sample was then injected onto a Acquity UPLC® Protein BEH SEC Column, 20θΑ, 1.7 μιη, 4.6 x 150 mm, Waters™ or an Agilent Bio SEC-3 4.6 x 300 mm size exclusion column [mobile phase 10 mM sodium phosphate buffer (pH 7.6), 100 mM KCl]. Both columns separate biomolecules in the range of 100-600 kDa; the Agilent Bio SEC-3 has a higher loading capacity and was used specifically for this reason for quantification of non-native TTR in Fig. 18F, Fig. 20 and Fig. 21. Aliquots of the samples either before or after SEC were conjugated with either Rhodamine-azide (tetramethylrhodamine azide, cat# 7130 Lumiprobe) or Diazo-Biotin-azide (cat# BP-22477, BroadPharm) using the copper catalyzed click reaction. For the click reaction, per 50 of sample, \ μΐ. of 5 mM azide containing probe (100 μΜ) was added to the sample and mixed, followed by the addition of 5 a 50:50 mix of 1 M CUSO4 and BTTP ligand. The reaction was then initiated by addition of 1 of sodium ascorbate (20 mg/mL in water) and incubated at 30°C for 1 hour with gentle mixing.

[00144] For sample sets in which non-native TTR labeling was directly compared by in- gel rhodamine fluorescence in different patient or control groups (e.g., asymptomatic V30M vs. V30M FAP or Pre vs. 12 mo Tafamidis) a standardized method was used. Plasma (47.5 μί) was added to 2.5 μί of lmM probe B-2 and rapidly mixed by pipetting up and down. The samples were then incubated overnight at 37°C (14-15 hours) and crosslinked in a 96- well plate as described above, run through a Bio-Spin® P-30 column, followed by injection onto an SEC column (Agilent Bio SEC-3 4.6 x 300 mm, run at 1 mL/min in 10 mM sodium phosphate buffer pH 7.6, 100 mM KC1). The high MW peak containing proteins that elute in the void volume and with a MW >200 kDa were collected (5-6 mL eluting peak). 200 μΐ. of this fraction was then taken and subjected to reaction with rhodamine azide via the copper catalyzed azide-alkyne click reaction. After completion of the click reaction, the proteins were precipitated with methanol/chloroform to remove any unconjugated rhodamine dye. The resulting protein pellet was resuspended in 50 μί of Laemmli buffer containing DTT and boiled for 5 minutes. 20 nL of the sample was then analyzed by SDS PAGE (4-12% BOLT Gel, Invitrogen™, run at 200 V for 30 minutes). To compare band intensities between multiple gels, a standard curve was loaded in each gel. The inventors used a 28 kDa recombinant protein (retroaldolase) labeled quantitatively at a single cysteine with an alkyne using a maleimide derivative (retroaldolase-alkyne). Thus for each sample set, the inventors separately conjugated rhodamine azide from an identical fluorophore stock to retroaldolase- alkyne and loaded varying concentrations of the rhodamine-labeled retroaldolase. This procedure is depicted in fig. 31. All gels were imaged on a Biorad ChemiDoc™ MP system and bands were quantified in ImageLab™ (Biorad).

[00145] For the biotin pull-down experiments, samples were first subjected to the CuAAC click reaction with N ₃ -Diazo-Biotin. After biotin conjugation, each sample was precipitated with methanol/chloroform and resuspended in 500 μί of 8 M Urea, 1 X PBS, 5 mM EDTA, followed by the addition of 140 μΐ. of 10% SDS. Following resuspension, the samples were then reduced by adding TCEP (10 mM final concentration) and incubated for 30 min at room temperature. The free sulfhydryls were then blocked by adding iodoacetamide to a final concentration of 12 mM and incubated at room temperature for 30 minutes in the dark. After reduction and alkylation, the samples were then added to 6 mL of PBS containing 100 of Streptavidin-conjugated agarose beads and incubated at room temperature with gentle rocking overnight. The biotin beads were then washed once with IX PBS, 0.5% SDS, 5 mM EDTA, 3 times with 2 M Urea, IX PBS, 5 mM EDTA, and 3 times with IX PBS. The samples were then eluted from the beads by incubation with 100 of dithionite containing solution (42 mM sodium dithionite, 0.5% SDS, IX PBS, pH ~7) for 30 min at room temperature. The samples were then run in a denaturing gel for western blot analysis or prepared for mass spectrometry analysis as described below.

[00146] For anti-TTR western analysis, the gel was transferred to nitrocellulose, blocked with Odyssey blocking buffer (Licor™), and probed with the anti-TTR antibody (rabbit anti- TTR, DAKO™) or a C-terminal TTR specific antibody made by immunizing mice against TTR5 ₀ -127. The membrane was then imaged using a Licor™ infared imager following the addition of an anti-rabbit or anti-mouse IR-800 secondary antibody (Licor™).

[00147] Quantitative Proteomics using TMT isobaric mass tags: Plasma samples from 3 unique individuals were run in parallel using the following scheme. Plasma (95 μί) was mixed with 5 μΐ. of either probe B-2 or probe B-2-Mut and incubated and crosslinked as described above. The plasma samples were then passed through a Bio-Spin® P-30 gel- filtration column equilibrated in 10 mM phosphate buffer pH 7.6, 100 mM KC1 and subjected to conjugation of N ₃ -Diazo-Biotin as described above. Probe-crosslinked proteins were enriched using streptavidin agarose as described above. The biotin-enriched eluted protein samples were then precipitated in methanol/chloroform and washed twice with 100% methanol. The air-dried protein pellets were then resuspended, reduced, acetylated and trypsin digested, and labeled with respective TMT-NHS isobaric reagents (Thermo Fisher) as described previously. The 3 plasma samples treated with either probe B2 or probe B2-mut were pooled, resulting in a total of 6 channels per run. MudPIT columns were prepared as described previously and LC-MS/MS analysis was performed using a Q-Exactive mass spectrometer with an EASY nLC 1000 (Thermo) LC pump. MudPIT experiments consisted of 5 min sequential injections of 0, 20, 50, 80, 100 % buffer C (500 mM ammonium acetate in buffer A) followed by a final step of 10 % buffer B (20% water, 80% acetonitrile, 0.1% formic acid v/v/v)/90% buffer C (95% water, 5% acetonitrile, 0.1% formic acid, v/v/v). Each injection was followed by a linear gradient from buffer A (95% water, 5% acetonitrile, 0.1% formic acid, v/v/v) to buffer B. Electrospray was carried out directly from the analytical CI 8 columns by applying a voltage of 2.5kV using an inlet capillary temperature of 275°C. Data- dependent acquisition of MS/MS spectra was performed as described before. Protein identification and quantification of TMT labeling intensities was carried out using the Integrated Proteomics Pipeline Suite (IP2, Integrated Proteomics Applications, Inc., San Diego, CA) as described previously. Global normalization of TMT intensities across the 6 channels was carried out within Census in IP2. Enrichment of a protein in the probe B-2- treated samples versus probe B-2-Mut-treated samples was calculated as the difference in log2 combined TMT intensities of the protein for a given patient. Enrichment differences were then averaged across the 3 patients and significance was tested in GraphPad Prism using unpaired t-tests assuming equal standard deviations across sample populations, followed by a multiple testing correction using a two-stage linear step-up procedure and a desired FDR of 5%.

Example 2

Β-β-strand of TTR labels misfolded TTR oligomeric aggregates prepared in

vitro

[00148] Transthyretin aggregation appears to form a spectrum of structures, including amyloid fibrils in buffers. The aggregation process is associated with a rough free energy landscape (Figure 1A, B), especially in vivo, probably due to the presence of extracellular holdase chaperones that kinetically stabilize misfolded and misassembled TTR structures, thus certain aggregate structures can be kinetically trapped. TTR aggregation in particular is very fast in vitro, although amyloid fibrils are not readily formed under aggregation reactions in buffers. Thus, the inventors hypothesize that misfolded oligomeric TTR structures would be less densely packed than native TTR or amyloid fibrils, allowing certain complementary peptides to integrate into misfolded TTR oligomer structures by docking and integrating into the structure at imperfection or defect sites (Figures 1A and IB). This hypothesis was tested by synthesizing peptides derived from the TTR primary sequence as candidate probes for detecting misfolded TTR oligomer structures.

[00149] An overlapping TTR peptide library comprising the 8 β-strands of TTR and the intervening loops or helix (Figure 1C) was synthesized. These peptides were labeled at the N-terminus with fluorescein and incubated with recombinant non-native TTR oligomers prepared in vitro from a monomeric version of TTR (M-TTR) (Figure ID). M-TTR comprises two mutations, one at each of the two distinct dimer interfaces (L110M, F87A) rendering M-TTR unable to assemble into a native tetramer, thus it can aggregate under physiological conditions because the typically slow step of tetramer dissociation has been eliminated. When incubated at neutral pH for one week, M-TTR undergoes conformational excursions allowing it to aggregate into non-native oligomers ranging in MW from 200-1000 kDa (Figure ID).

[00150] After overnight incubation of each candidate TTR-derived peptide probe with the non-native M-TTR oligomers, peptide incorporation was assayed by Native PAGE visualized by fluorescein fluorescence and by Size Exclusion Chromatography (SEC) monitored by fluorescein fluorescence. Of the 18 fluorescent peptides that were initially evaluated, only the peptide derived from the B β-strand of TTR, hereafter called peptide probe B-l, bond to and apparently incorporated into the misfolded M-TTR oligomers with high efficiency (Figures ID and E). That probe B-l remains bound after gel filtration chromatography suggests that it dissociates very slowly from the oligomer, thus it is likely to have integrated into a β-sheet rich structure at a defect site (Figures 1A and IB).

[00151] The same approach was used to assess whether probe B-l incorporated into TTR oligomers made from TTR sequences other than full-length TTR. Since the B β-strand is located near the N-terminus, the inventors tested TTR oligomers made from TTR5 ₀ -127, which lacks the B β-strand. This TTR fragment is deposited in cardiomyopathy and some polyneuropathy patients. TTR5 ₀ -127 is highly oligomerization prone, affording TTR oligomers based on native gel analysis and atomic force microscopy (fig. 22). Despite differences in oligomer size and sequence, again probe B-l is the only peptide of 18 that incorporates significantly into these oligomers (fig. 23).

Example 3

Probe B-l is a selective for identifying misfolded TTR oligomers

[00152] Control experiments demonstrate that probe B-l is selective for binding to and integrating into the structure of misfolded TTR oligomers. Upon incubation of probe B-l with native WT TTR tetramers, or freshly purified V30M or T119M or L55P or T60A or A25T TTR homotetramers comprising disease-associated TTR subunits, no significant labeling was observed, indicating that probe B-l is selective for non-native aggregate forms of TTR over natively folded TTR (Figure 2A, 16A). The highly destabilized A25T tetramer readily dissociates under physiological conditions and forms high MW TTR oligomers within hours, and as such, probe B-l intensely labels these non-native TTR oligomeric aggregates (Figure 2A). After correction for fluorescence quenching (fig. 23), it was found that MTTR oligomers undergoing active aggregation more readily incorporate probe B-l compared to MTTR that has been oligomerizing for more than 15 days (Fig. 16B), in a stoichiometry of ^¾ 1 : 1 peptide probe:MTTR monomer comprising the oligomer (fig. 23). These results suggest that a fundamental structural commonality, appearing to involve residues 50-127, exists between the MTTR, TTR5 ₀ -127 and A25T non-native TTR oligomers that allow probe B-l binding and apparent structural integration. This structural motif is not present in native TTR tetramers or folded MTTR.

[00153] Next, probe B-l was used on salivary gland biopsy from a FAP patient and a sectioned human heart from a TTR FAC patient, revealing that unlike Congo red, probe B-l does not bind to or integrate into amyloid. To test whether probe B-l was able to incorporate into TTR amyloid fibrils, salivary gland biopsies of a V30M FAP patient and a control were stained with a biotin labeled version of probe B-l (B-l-Biotin). The localization of probe B- 1 -Biotin was compared with i) Thiofiavin T fluorescence (an amyloid selective fiuorophore) and ii) anti-TTR antibody localization. The amyloid fibrils in the salivary gland were found to be localized around glandular acini as revealed by Thioflavin T fluorescence (Fig. 16C (top left panel; white arrowheads) and fig. 24, and the anti-TTR antibody confirmed these are TTR amyloid (Fig. 16C; third panel from top left). In contrast to this amyloid localization, probe B-l-Biotin was found inside the glandular acini of both patient and control (Fig. 16C and fig. 24, white asterisks), revealing that the peptide does not bind to or integrate into the amyloid fibrils present within tissue, and that the probe B-l-Biotin fluorescence signal results from non-specific binding to other proteins inside the glandular cells.

[00154] Next, it was assessed whether probe B-l incorporated into aggregates made from proteins other than full-length TTR. Since the B- ?-strand is located near the N-terminus, the inventors tested aggregates made from TTR52-127, a highly aggregation prone fragment that has been implicated in TTR related cardiomyopathies and polyneuropathies, notably a sequence lacking the Β-β-strand. The entire TTR peptide probe library was screened against the TTR52-127 oligomeric aggregates, and only probe B-l incorporated into TTR52-127 aggregates. The TTR52-127 fragment is highly aggregation prone and forms soluble aggregates that are morphologically distinct from M-TTR oligomers. Despite differences in size, morphology, and sequence, probe B-l was the only peptide that incorporated significantly into these aggregates (Fig. 8). These results suggested that there exists a fundamental structural commonality between all of these non-native TTR oligomeric forms, which allowed probe B-1 to recognize these aggregates while excluding probe B-1 incorporation into native TTR. This structural motif appeared to involve residues 52-127 common to all aggregates. In one embodiment, probe B-1 may integrate into WT TTR aggregates made at pH 4.4.

[00155] Notably, it was found that M-TTR non-native TTR oligomers and TTR52-127 misfolded oligomers actively aggregating more readily incorporate probe B-1, relative to oligomers that have been aged (> 2 weeks for M-TTR neutral pH aggregation reaction and > 1 day for the TTR52-127 aggregation reaction), at which point the defect sites appear to diminish perhaps owing to conformational conversion (Fig. 9). Finally, time course experiments indicate a generally slow rate of incorporation of probe B-1 into M-TTR oligomers that have been aggregating for 7 d, reaching saturation by 12-24 h at the indicated concentrations (Fig. 9). These attributes, combined with the observation that the probe B-l- misfolded TTR oligomer complex is resistant to dilution by gel filtration (Figure IE) qualitatively characterizes probe B-1 as a slow, very tight, and selective binder of non-native TTR among other conformations-the off-rate appears to be very slow based on the chromatography experiments. Additionally, the probe B-1 was screened against Abetal-42 ADDL oligomers. The probe B-1 exhibited no binding to the monomer, dimer and trimer species present, and minimal binding to higher order aggregates which is consistent with prior reports (Fig. 10).

Example 4

Probe B-1 structure-activity relationship suggests that probe B-1 is integrating

into a cross β-sheet structure

[00156] An alanine scan within the probe B-1 sequence was performed. From these experiments a clear structure activity relationship emerged - every other residue was important in the core of probe B-1 (AINVAVHVFR) (SEQ ID NO: 2) for its incorporation into misfolded TTR oligomers, namely the 2 ^nd , 4 ^th , 6 ^th and 8 ^th residues. The apparent requirement for every other residue being a β-branched amino acid is consistent with the peptide binding in a β-strand conformation or integrating into a cross β-sheet structure harboring a defect site (Figure 1A). Alanine substitution of the three central valine residues (V28, V30, V32 numbered according to the WT sequence) significantly compromise recombinant aggregate integration, while the intervening position Ala substitutions were determined to have only a minor effect on probe binding/integration to misfolded TTR oligomers as shown in Figure 2B. In addition to the substitutions shown in the figure, other mutations of probe B-l were made by replacing the intervening positions with lysine or tyrosine. These include Lys substitutions for the 3 ^rd or the 7 ^th residue and Tyr substitution for the 9 ^th residue. It was found that these replacements all had a minimal or no effect on peptide incorporation. Furthermore, truncation of the peptide probe B-l from the N- and C-termini identified the sequence VAVHVF (SEQ ID NO: l) as a minimal binding/integration competent sequence, although the truncated peptide incorporates to a lesser extent (-90% less) in comparison with the original probe B-l sequence itself (Fig. 11).

Example 5

Probe B-l selectively labels the misfolded TTR oligomers in ATTR patients

[00157] A major goal of this work was to develop peptide-based probes that are able to detect misfolded forms of TTR in relevant biological contexts, such as in the blood, plasma or cerebrospinal fluid. In the case of TTR polyneuropathy or cardiomyopathy patients, the inventors have demonstrated that circulating tetrameric TTR dissociates, and monomeric TTR misfolds and misassembles into soluble oligomers or other aggregate types. The inventors tested whether these aggregates are present in plasma of patients with symptoms of disease and whether they can be used as early diagnostic markers. Furthermore, the development of these types of probes also allowed them to determine if the lowering of non- native TTR oligomers may correlate with clinical outcome upon disease modifying therapy treatment.

[00158] The inventors first evaluated whether probe B-l could label M-TTR oligomers that were added to the human plasma of a healthy blood donor (Figure 2C). In this context, probe B-l selectively labels the recombinant non-native M-TTR oligomers (Figure 2D & E). Moreover, a standard curve exhibits a linear probe B-l response with misfolded oligomeric TTR concentration (Fig. 12). Other proteins are labeled to a lesser degree, including serum albumin, which is not unexpected, as albumin is known to bind to short, relatively hydrophobic peptides. Notably, a similar SAR trend was observed when probe B-l alanine mutants were added to plasma containing non-native M-TTR oligomers (Figure 2F).

[00159] Based on these ex vivo results, the inventors tested whether this probe would be able to specifically label circulating non-native oligomeric TTR aggregates in patient plasma - if they are present (Figure 3 A). Probe B-l was incubated overnight with plasma samples from ten symptomatic V30M FAP patients, ten asymptomatic V30M mutation carriers, and two patients with wild-type TTR cardiomyopathy. All 12 patients (10 V30M FAP and 2 WT cardiomyopathy) had symptoms defining the onset of disease at the time the blood samples were collected. Upon size exclusion or gel filtration chromatography of each patient plasma sample after incubation with probe B-l, a statistically significant increase in the fluorescence peak eluting between 800-1200 (the misfolded TTR oligomer peak exhibiting a MW of 150-650 kDa) relative to the healthy control samples (Figure 3B and C, p-value = 0.0003) was found. These observations suggest that soluble non-native TTR oligomers exist in plasma derived from patients, but is not present in the control plasma samples. The oligomeric peak does not react with any of the fluorogenic native TTR probes used herein, further evidence that the TTR in this peak is misfolded (Liu et al, J. Am. Chem. Soc. 137, 11303, 2015; and Suh et al, J. Am. Chem. Soc. 135, 17869, 2013). Probe B-l is the only TTR-derived peptide probe that integrates into the TTR oligomers in patient plasma (Figure 3D). These results also demonstrate the potential utility of probe B-l as a diagnostic probe. It is reasonable to expect that the soluble misfolded TTR oligomer peak in plasma could contain holdase chaperones like ERdJ3, or Clusterin, and possibly glycosaminoglycans and other components.

Example 6

Structure activity relationship of probe B-l is analogous in patient plasma

[00160] The SAR trend of the probe B-l alanine mutants added to patient plasma in comparison to being added to control plasma to which recombinant non-native M-TTR oligomers had been added are very similar (Figure 3E and Figure 2F). Individual substitution of the three core valines (V28, V30 and V32) by Ala in the probe B-l peptide analogs reduces the intensity of 800-1200 gel filtration peak corresponding to the non-native TTR oligomers in patients by 70% (relative to probe B-l). Substitution of 126 or F33 by Ala also reduced the 800-1200 gel filtration peak obtained from incubation of the peptide with polyneuropathy and cardiomyopathy non-native TTR oligomers in plasma (by ~ 60%). This is consistent with what was observed when these Ala mutants of probe B-l was incubated with M-TTR oligomers in buffer or when M-TTR oligomers are added to plasma of healthy donors prior to incubation with the peptide probe B-l analogs (Figure 2F). The substitution of N27 and H31 by Ala gave a higher 850-1200 μΐ. gel filtration signal in both patient plasma and with M-TTR oligomers spiked into healthy plasma; however the signal found in healthy controls (with no oligomers added) was also higher with these two probe B-l peptide analogs. The N27 and H31 probe analogs are not useful as probes, because the difference between the intensity of the 850-1200 gel filtration peak in patients vs. healthy controls became statistically non-significant, suggesting that the higher control plasma signal is coming from binding to non-specific high-molecular weight proteins or protein complexes. These results suggest that the B-1 peptide probe could be recognizing circulating TTR- derived oligomers in patients that are very similar to the non-native M-TTR oligomers prepared in vitro, which have not have been detected previously.

Example 7:

Photo-crosslinker identifies non-native oligomeric TTR as the target of probe

B-1 in FAP patient plasma

[00161] To distinguish between probe B-1 binding to naked non-native TTR oligomers in FAP patient plasma versus non-native TTR oligomers interacting with holdase chaperones and potentially other plasma protein binding partners, a diazirine functional group and an alkyne handle was incorporated into probe B-1, affording probe B-2 (Fig. 18 A). Upon irradiation at 355 nm, the diazirine forms a highly reactive short-lived (-100 ns) carbene that inserts into proximal bonds, resulting in covalent conjugates with the target protein(s) and potentially other macromolecules. After incubation of probe B-2 with plasma samples, the samples were irradiated and rhodamine-azide was covalently attached to the alkyne handle using a denaturing copper catalyzed alkyne-azide cycloaddition or 'click' reaction (CuAAC) to render the conjugates fluorescent. The advantage of probe B-2 over probe B-1 is that after photocrosslinking, probe B-2 is covalently attached to the proteins it initially binds to, thus denaturing and reducing SDS PAGE can be used to identify the proteins bound by probe B-2, including initially aggregated non-native TTR (Fig. 18B). In contrast, non-denaturing separation methods like SEC must be used to preserve the non-covalent probe B-1 interactions.

[00162] Probe B-2 selectively labels MTTR oligomers, both in buffer (fig. 30) and when oligomers were added to the plasma of healthy donors, but does not label natively folded tetrameric TTR that is also present in μΜ concentrations within plasma (Fig. 18C, middle panel, magenta box) indicated by the intense rhodamine fluorescence signal at the TTR monomer MW, and absence in the control lanes (cf. lanes 1 and 2 of the B-2 Rhodamine data). Thus probe B-2 displays a high degree of conformational selectivity for non-native TTR oligomers, as does probe B-1. Control experiments with peptide probe B-2C, lacking the diazirine functional group, exhibited minimal TTR labeling (Fig 18C; middle panel, lanes 3 and 4), indicating that labeling is dependent on the presence of the diazirine cross-linker, facilitating denaturing SDS-PAGE separation and analysis (Fig. 18C).

[00163] Probe B-2 was incubated with FAP patient plasma. These samples exhibited the high MW SEC fluorescence signal upon incubation with probe B-1. After photocrosslinking and clicking on rhodamine, a band that migrates equally to that of monomeric TTR (13.5 kDa) is covalently labeled by probe B-2 in the denaturing SDS-PAGE of FAP patient plasma, but minimally in the plasma derived from a healthy donor (Fig. 18D, middle panel, magenta box), again demonstrating that probe B-2 does not form a complex with the natively folded TTR tetramer prior to photocrosslinking. Notably, other higher MW proteins are labeled by probe B-2 differentially between patients and controls, as revealed by the SDS-PAGE readout (Fig. 18D, middle panel).

[00164] To identify the proteins present in the high MW SEC fractions, representative plasma samples were incubated with probe B-2, photocrosslinked, and then these plasma samples were subjected to non-denaturing SEC fractionation prior to performing a denaturing rhodamine click reaction on each chromatographic fraction, followed by denaturing SDS- PAGE (Fig. 18E summarizes workflow). In V30M FAP plasma, TTR is clearly present in the high MW SEC fractions (3-5; i.e. 800-1200 μΐ,) based on SDS-PAGE analysis (Fig. 18F, middle panel, magenta box). TTR observed as a monomer in SDS PAGE in the rhodamine channel was in a more than 200 kDa MW complex, because only TTR in the "high MW fractions" (i.e., 3-5) reacted with covalent probe B-2. In contrast, a healthy donor plasma sample incubated with probe B-2 and irradiated does not crosslink with native WT TTR (Fig. 18F; right side middle panel), despite the fact that TTR is present in nearly all fractions, as ascertained by using an anti-TTR antibody (Fig. 18F, bottom panel, dark green box). Thus, probe B-2 only labels high MW non-native TTR oligomers in the patient plasma and not native TTR in healthy donor plasma, demonstrating the selectivity of probe B2 for non-native oligomeric TTR in plasma.

[00165] The intensity of the B-2-TTR conjugate band was quantified from SDS-PAGE of the high MW plasma fraction by densitometry in a larger subset of V30M FAP patients, asymptomatic V30M carriers, and healthy donor controls. In the control groups, only minimal labeling of TTR is observed, whereas in the V30M FAP patient group, significantly more non-native TTR is labeled (Fig. 18G & fig. 31; Table 1).

[00166] Probe B-2 cross-linked plasma proteins were subjected to a click reaction with biotin, and subsequently the probe B-2 crosslinked proteins were affinity purified in patient vs. control plasma. TTR is clearly identified in the eluted fractions by SDS-PAGE visualized by anti-TTR western blot, and is more prominent in the patient samples (Fig. 18H). Control experiments show that the B-peptide sequence itself does not cross-react with the anti-TTR antibody used (Dako Inc., #A0002) (Figure 13). Moreover linear epitope mapping identifies the C-terminus of TTR as the primary epitope that the Dako TTR anibody targets. Thus the anti-TTR antibody is recognizing an antigen other than the B-peptide sequence (fig. 32).

Table 1 : Sample age and demographics for the non-native TTR detection by the B-2 SDS PAGE assay presented in Fig. 18H, Fig. 20, and Fig. 21.

Example 8

Cleaved TTR is identified in a fraction of patients

[00167] Notably in some patient plasma samples treated with probe B-2 and irradiated, labeling of a lower MW band (~9 kDa) is observed. This is the expected MW of the C- terminal TTR fragment found to comprise amyloid fibrils in a subset of TTR amyloidosis patients, i.e. TTR52-127 (8666 Da) (Figs. 4C, 4F and Fig. 14). In a previous histopathologic study of FAP and WT-cardiomyopathy patients, this fragment was identified in all cardiac tissue biopsies from WT-TTR cardiomyopathy patients, while in the FAP patients a mixture of cleaved and full-length TTR was detected in the amyloid fibrils that were extracted (Arvidsson et al, PLoS ONE 10: e0143456, 2015; and Bergstrom et al, J. Pathol. 206, 224, 2005). It is unclear from these studies, whether TTR is cleaved prior to deposition or within the tissue environment after deposition. Upon biotin conjugation, streptavidin enrichment and western blotting, this fragment is identified to be TTR. This suggests that TTR is cleaved in the blood, and that fragmented TTR may in fact be circulating.

Example 9

TTR labeling bv B-2 correlates with high MW SEC signal

[00168] The inventors observed that TTR labeling by B-2 strongly correlates with the high MW SEC signal. The results indicate that both probe B-2 photo-crosslinking and probe B-1 fluorescence chromatography can be used as general diagnostic for ATTR pathologies. Specifically, a standardized procedure was used where patient or control plasma was incubated with probe B-2. This was followed by collection of the high MW fraction using size exclusion chromatography, rhodamine labeling, and separation by reducing SDS PAGE. The labeling of a subset of plasma samples by probe B-2 was then quantified by densitometry (Figure 5A). As shown in the figure, probe B-2 labeling of TTR in SDS PAGE correlates strongly with the integrated area of the size exclusion chromatography fluorescence peak that results from incubating patient plasma with probe B-1 (Figure 5B; correlation coeff = 0.85, p-value = 0.0052). These results as quantified in Fig. 5 demonstrate the utility of the either the probe B-2 photo-crosslinking approach and/or the probe B-1 UPLC-SEC fluorescence chromatography method as a potential diagnostic to distinguish symptomatic patients from controls (asymptomatic V30M mutation carriers, and healthy WT TTR donors).

Example 10

Probe B-1 selectively differentiates FAP patient samples from controls

[00169] Native PAGE and SEC was employed to discern whether probe B-1 could label exogenous MTTR oligomers that were added to the plasma of a healthy donor. In this context, probe B-1 selectively labels the non-native MTTR oligomers and exhibits a linear response with misfolded oligomeric TTR concentration based on SEC analysis (fig. 25). Notably, an Ala-scan structure-activity relationship (SAR) with probe B-1 analogs added to plasma containing non-native MTTR oligomers (fig. 26) was similar to that observed with non-native MTTR oligomers in buffer (Fig. 1), addressing selectivity even in this complex biological fluid.

[00170] Probe B-1 was incubated with plasma from symptomatic Portuguese and Japanese V30M FAP patients, asymptomatic Portuguese V30M mutation carriers, and healthy controls. All patients had symptoms defining disease onset at the time the blood samples were collected and most patients had a Neurological Impairment Score of the lower limbs (NIS-LL) of less than 20 points, reflecting early stage FAP. By both SEC and native PAGE analysis, probe B-1 labeled the high MW fraction in the FAP patients (Fig. 17A and B). In contrast, only minimal labeling was observed in the control groups. SEC analysis of plasma afforded a statistically significant increase in the high MW fluorescence peak eluting between 800-1200 in the FAP patients relative to the control groups (Fig. 17C). Identical results were obtained using a B-peptide wherein the fluorescein substructure is replaced by disulfoCy5, showing that use of a less environmentally sensitive fluorophore makes no difference in this context (fig. 27).

[00171] The SAR of the probe B-1 alanine mutants with V30M FAP patient plasma shows the same trend as that exhibited when the probe B-1 alanine mutants were added to control plasma to which MTTR oligomers were added (fig. 28). Substitution of any of the core β- branched amino acids eliminates the fluorescence intensity of the 800-1200 gel filtration peak. TTR-based peptide probes from the eight β-strands (Fig. 1C) were reevaluated using FAP patient plasma, and only probe B-1 was found to exhibit fluorescent intensity in the high MW gel filtration peak (800-1200 μί; fig. 29).

[00172] Collectively, these results demonstrate that probe B-1 recognizes circulating misfolded TTR oligomers in patients that have not been detected previously. It is likely that the "TTR Oligomer-probe B-1" peak in plasma could also comprise holdase chaperones like ERdJ3 and clusterin, and possibly glycosaminoglycans and other macromolecular components.

Example 11

Quantitative proteomics identifies the targets of the B-2 probe beyond non- native TTR oligomers

[00173] The inventors next sought to identify other targets of the B-2 peptide probe. They hypothesized those circulating extracellular chaperones, which are present in high concentration within human plasma, could be a potential target of probe B-2. They tested for the presence of two protein candidates with known holdase activity, Clusterin and Haptoglobin, which are in top 20 most abundant plasma proteins. Clusterin is a multifunctional homodimeric 37 kDa sulfated glycoprotein that has been found to be co- deposited with amyloidogenic proteins in histopathology studies. Its variants have notably been positively linked in GWAS studies to neurodegenerative pathologies - most notably in Alzheimer's. Haptoglobin is also a multifunctional high abundance plasma protein that has been shown to have chaperone activity and whose alpha subunit is similar in MW weight to TTR (9 and/or 18 kDa depending on the haplotype).

[00174] Upon blotting of B-2 labeled patient or control plasma for TTR, the inventors sequentially blotted for Clusterin and the Haptoglobin alpha chain. No overlaying signal was observed for the Haptoglobin alpha chain, indicating that this protein is likely not a target of probe B-2 (Fig. 15). However, in both the anti-Clusterin and B-2 rhodamine channels an intense band at 37 kDa overlays and in B-2 pull-down experiments Clusterin is identified indicating that Clusterin is a target of B (Figure 6). This is consistent with the results from a previous reported Clusterin pull-down experiment (Humphreys et al., J. Biol. Chem. 274, 6875, 1999).

[00175] Furthermore, upon comparison of patient and control groups, labeling of Clusterin by B-2 notably is suppressed in patients (Figure 4c). The same suppressed labeling of Clusterin by B-2 is observed when recombinant M-TTR oligomers are added to healthy control plasma, suggesting that non-native TTR and probe B-2 display competitive binding to Clusterin (Figure 4b). Thus, labeling of Clusterin may also be useful as a diagnostic signature of TTR amyloidosis.

[00176] Unbiased proteomics experiments were performed to compare the relative abundances of the probe B-2 target protein conjugates in plasma samples from 3 Portuguese V30M FAP patients, 3 Portuguese asymptomatic V30M carriers, and 3 healthy donors (Fig. 19A summarizes the workflow). Probe B-2 was incubated with the plasma samples overnight, photocrosslinked and the conjugates were affinity purified by clicking on biotin to the B-2 alkyne handle. As a control probe, an identical diazirine and alkyne containing B peptide analog harboring a single alanine substitution (B-2-Mut, AINAAVHVFR, V28A Fig. 1G as well as fig. 26 and 28) was used, which eliminated labeling of MTTR oligomers. Rhodamine gel labeling experiments confirmed that probe B-2-Mut does not label non-native TTR oligomers in FAP patients (Fig. 19B, rightmost panel, magenta box). After affinity purification, each sample was digested with trypsin, and the tryptic fragments were labeled by one of the six unique isobaric mass tags (TMT tags, amine reactive). The 6 samples (e.g. the 3 unique V30M FAP patients treated with B-2 and B-2-Mut) were then combined and subjected to MudPIT LC-MS/MS analysis and the relative abundances of the tryptic peptides in each of the six samples were quantified by the intensity of the unique fragments in the MS2 spectra. Proteins of interest are expected to be labeled by probe B-2, but not the probe B-2-Mut. In other words, the intensity of the identified peptides in the MS2 spectra will be higher in the probe B-2 treated TMT channels, relative to the probe B-2-Mut TMT channels.

[00177] In the FAP data set, 99 unique proteins were identified by the affinity purification mass spectrometry approach, the highest intensity protein being serum albumin, as expected because it binds hydrophobic peptide probe B-2 and is by far the most abundant protein in human plasma (Fig. 19C). For the high abundance plasma proteins, the spectral counts of the identified proteins correlate with their expected plasma concentration (fig. 33). Importantly, however, when the protein list is sorted by the intensity ratio of probe B-2 to probe B-2-Mut, the most "B-2 enriched" proteins are TTR, several apolipoproteins including ApoE (36 kDa), clusterin (37 kDa), and alpha-2-macroglobin (-180 kDa), i.e., they have the highest intensity ratio of probe B-2 to probe B-2-Mut (Fig. 19D and E). Notably, the majority of the proteins that are most confidently identified by proteomics herein have been previously found in amyloid tissue biopsies from FAP patients, suggesting that these proteins may circulate with non-native TTR.

[00178] A clear proteomic signature emerges that distinguishes FAP patients from healthy donors and asymptomatic carriers. In the controls, the maj ority of probe B-2 targets identified in the FAP patients are also identified, including TTR (Fig. 19C). The enrichment ratio for TTR was greatest for FAP patients followed by asymptomatic carriers, followed by healthy controls exhibiting no enrichment (probe B-2 : probe B-2-Mut ~ 1) (Fig. 19F). In contrast, the ratios for the circulating interacting proteins, namely clusterin (a holdase chaperone), ApoE (transports lipoproteins, fat soluble vitamins and cholesterol in blood), and vitronectin (functions in hemostasis and modulating cell adhesion) display an inverse trend, i.e., the probe B-2 : probe B-2-Mut ratio is lowest for the FAP patients (Fig. 19F). This suggests that non-native TTR oligomers may be interacting directly with these proteins, displacing probe B-2. Alternatively, under the concentrations employed, non-native TTR oligomers, when present, could out-compete the available probe B-2 from binding to these other protein targets. Nonetheless, the biochemical and proteomics experiments described thus far fully validate non-native oligomeric TTR as a clear target of the B-2 probe, along with several TTR amyloid associated proteins.

Example 12

Correlation of misfolded TTR oligomers with symptoms and treatment

[00179] The inventors observed that circulating misfolded transthyretin oligomers in plasma of familial amyloid polyneuropathy patients correlate with early stages of clinical symptoms and decrease upon tafamidis treatment. As shown in Figure 3C, probe B-l did not detect oligomers in plasma of mutation carriers that are asymptomatic, i.e., no symptoms or signs suggestive of polyneuropathy or any other organ involvement. In order to determine if probe B-l is able to detect patients very early in their disease course, samples from patients with very few neurological symptoms (defined as NIS < 10) were analyzed. When the levels of oligomers detected by the B-l probe with NIS were compared, a moderate correlation was seen, with higher levels of oligomers detected in patients with more symptoms (r ² =0.5013, Figure 7A). Similar results were obtained with probe B-2.

[00180] Tafamidis is a transthyretin kinetic stabilizer that is regulatory agency approved in Europe and Japan for the treatment of FAP. The levels of non-native oligomers present in the plasma of tafamidis treated patients were investigated using probe B-l. These patients have been treated with tafamidis for at least 12 months and are considered clinical responders, i.e., with progression of the neurological impairment score scale less than 2 points (Suanprasert et al, supra). The inventors found that these TTR amyloidosis patients exhibit a reduction in the high molecular weight peak that is labeled by B-l after at least 12 months of tafamidis treatment. Figure 7B shows the chromatograms of one of these typical responder patients (before treatment and after 12 months of tafamidis treatment), labeled with B-l . The amount of non-native TTR labeled by B-l drops by about 50% after 12 months of treatment.

[00181] Covalent probe B-2 was also used to analyze the levels of non-native TTR oligomers in the plasma of 15 Portuguese V30M FAP patients at the time of diagnosis and after 12 months of tafamidis (20 mg daily) administration (average NIS-LL change = -0.1). The majority of these patients were diagnosed early in the course of the disease with NIS-LL scores of < 10 and are considered to be clinical responders (i.e., annual progression of the neurological impairment score of less than 2 points). Labeling of non-native TTR in the high MW SEC fraction by probe B-2 was reduced or unchanged in 13 of 15 patients, and increased in 2 patients (Fig. 20A and B). On average in the 15 patients analyzed, non-native TTR labeling decreased by 1.8 fold after 12 mo of tafamidis treatment. Analysis of more patients on different doses of tafamidis will be required to discern whether covalent probe B- 2 has potential as a response-to-therapy marker.

[00182] Furthermore, the inventors tested 7 Japanese V30M FAP patients who underwent liver transplant-mediated gene therapy several years earlier, eliminating the presence of the V30M protein in the circulatory system and slowing the progression of FAP. In these samples, no detectable level of non-native TTR was observed (Fig. 20C). Example 13

Generally

[00183] Transthyretin (TTR) is a 127-amino-acid β-sheet-rich tetrameric protein that is predominantly secreted into the blood by the liver. Local production of TTR by the choroid plexus and the retinal epithelium accounts for the smaller quantities of TTR in the cerebrospinal fluid (CSF) and the eye, respectively. Natively folded tetrameric TTR circulating in blood, CSF, and in the eye of healthy individuals is known to function mainly as a transporter of vitamin A and thyroxine. This natively folded TTR tetramer can slowly dissociate into monomers, that can subsequently misfold, enabling TTR aggregation, a process driving the dysfunction and ultimately the loss of post-mitotic tissue in a heterogeneous group of diseases collectively known as the TTR amyloidoses.

[00184] Approximately 120 amyloidosis-associated TTR mutations have been described so far; the autosomal dominant inheritance of one of these mutations leads to the incorporation of mutant subunits into a TTR tetramer otherwise composed of wild-type subunits, causing heterotetramer destabilization, i.e., faster TTR tetramer dissociation kinetics and/or the accumulation of higher quantities of misfolded aggregation-prone monomers. The hereditary TTR amyloidoses are systemic amyloid diseases that can present with a variety of clinical phenotypes. It is currently established that patients with certain mutations present predominantly with a cardiomyopathy (e.g., V122I), while other mutation carriers exhibit predominant involvement of the peripheral nervous system (e.g., V30M, associated with Familial Amyloid Polyneuropathy or FAP), especially in high prevalence areas such as Japan and Portugal. Although the initial disease phenotype depends partially on the inherited TTR sequences, variability in clinical presentation is seen between patients with the same mutation and even within the same kindred, and some patients present with marked involvement of other less commonly involved organs, such as the eye (e.g., vitreous opacities and glaucoma), the central nervous system (e.g., stroke and dementia) or the kidney (e.g., nephrotic syndrome and chronic renal insufficiency). This poorly understood phenotypic variability (or tissue tropism) seen in the hereditary TTR amyloidoses, poses a significant diagnostic challenge. These patients often present first to different clinical specialties, with initial symptoms that can mimic a number of more common diseases, and there is currently no single diagnostic method that is non-invasive and easy to apply to diagnose the TTR amyloidoses. Genotyping and amyloid fibril detection in tissues, combined with confirmed organ damage detected by echocardiography and/or neurophysiological assessment, are generally considered the diagnostic gold standard. Recent advances in imaging methods using bone scintigraphy show promising results in early diagnosis of the TTR amyloidoses presenting as a primary cardiomyopathy, potentially avoiding the need for invasive diagnostic strategies. Such an approach is unavailable for the neurological presentation, and as a direct result, diagnosis is often made later in the course of the disease. This is problematic, as currently available therapeutic strategies— liver transplant-mediated gene therapy or the use of pharmacologic kinetic stabilizers (tafamidis and diflunisal)— has proven to be more effective when used early in the course of FAP, highlighting the need for a non-invasive early diagnostic method.

[00185] As with other more common amyloid diseases (e.g., Alzheimer's disease, Parkinson's disease), the mechanism(s) by which TTR aggregation or amyloidogenesis leads to organ dysfunction is not well understood. Although there is compelling genetic and pathological evidence that the process of amyloidogenesis is the cause of the TTR amyloidoses, the insoluble cross- -sheet amyloid fibril burden does not seem to correlate with clinical manifestations based on the observation that positive clinical responses to kinetic stabilizer therapy and/or liver transplant in TTR cardiomyopathy patients do not correlate with amyloid clearance (based on heart wall thickness measurements). The same has been observed with disease-modifying therapies in light chain amyloidosis (another systemic amyloid disease). Moreover, there is evidence from cell-based toxicity studies that soluble misfolded TTR oligomers are more toxic than amyloid fibrils, although the relevance of these short-term in vitro toxicity studies to degenerative diseases that manifest over months to years remains unclear.

[00186] In one embodiment, in the present disclosure, the inventors developed reliable probes for each structure in the spectrum of aggregate structures that exists in patients. Determining which structures correlate with symptom development by studying asymptomatic mutation carriers and monitoring changes in the concentration of aggregate structures in patients in response to drug treatments that clinically halt amyloid disease progression provided an insight into structure-proteotoxicity relationships that drive amyloid diseases. In one embodiment, the goal of such an activity was to eliminate structures that do not correlate with symptom development and/or with clinical response to therapy, so as to produce a list of misfolded species that may drive loss of post-mitotic tissue (proteotoxicity) in human amyloid diseases.

[00187] Thus, the present disclosure provides probes that can selectively and/or specifically detect circulating non-native TTR structures that form in addition to amyloid fibrils. In one embodiment, the present disclosure provides peptide-based probes that selectively integrate into the structure(s) of non-native TTR oligomers prepared in vitro and notably integrate into apparently similar structures circulating in the blood of hereditary TTR amyloidosis patients, specifically those with predominant neurological or mixed peripheral nervous system and cardiac phenotypes, but not in patients with primarily cardiac phenotypes. This approach of quantifying misfolded TTR oligomers in patient plasma could be useful not only for aiding physicians in point-of-care diagnosis and for following the response to particular therapies, but also for understanding the aggregate structure- proteotoxicity relationships driving the TTR amyloidosis.

Example 14

Proteolyzed TTR is identified in some FAP patient plasma samples.

[00188] In a few patient plasma samples treated with covalent probe B-2, labeling of a lower MW TTR band (-9-10 kDa) is observed in the high MW SEC fraction (Fig. 20A and fig. 34A, e.g., patients 5 and 11). This fragment is slightly larger than the expected molecular weight of the C-terminal TTR fragment (TTR5 ₀ -127, 8666 Da) found to comprise amyloid fibrils deposited in tissue in a subset of TTR cardiomyopathy patients. In a histopathologic study of FAP and WT TTR cardiomyopathy patients by Westermark and colleagues C- terminally cleaved TTR was identified in all cardiac amyloid fibril biopsies (WT TTR cardiomyopathy patients), whereas a mixture of cleaved and full-length TTR was detected in the deposited amyloid fibrils that were extracted from FAP patients. (J. Bergstrom et al Amyloid deposits in transthyretin-derived amyloidosis: cleaved transthyretin is associated with distinct amyloid morphology. J. Pathol. 206, 224-232 (2005)); (S. Arvidsson et al, Amyloid Cardiomyopathy in Hereditary Transthyretin V30M Amyloidosis - Impact of Sex and Amyloid Fibril Composition. PLoS One 10, (2015)). It was unclear from these publications whether TTR is cleaved prior to deposition or within the tissue environment after deposition.

[00189] These patient plasma samples were further probed by western blot using a C- terminus-specific TTR antibody (fig. 34B). In patients that exhibit labeling of this band by B- 2 (e.g., patients 5 and 11) a clear band is observed at approximately 9-10 kDa in SDS-PAGE by western blot analysis. Control experiments in which a protease inhibitor cocktail was added immediately after thawing the plasma samples showed no difference in the intensity of the TTR cleavage band, demonstrating that active proteolysis during peptide probe incubation is likely not responsible for the observed TTR proteolysis (fig. 34C). Collectively, the data outlined in Fig. 20 and fig. 34 suggest that C-terminally proteolyzed TTR is circulating in the blood of a subset of patients, although the possibility that the cleavage of TTR occurred during the period of blood collection and shipment could not be eliminated. Extensive experimental follow-up will be necessary to distinguish between these possibilities.

Example 15

Non-native TTR is detected in 3 additional polyneuropathy genotypes and not

detected in cardiomyopathy related genotypes.

[00190] Further scrutiny of whether covalent probe B-2 can detect non-native TTR oligomers in plasma of genotypes other than those harboring the V30M FAP mutation was undertaken (Fig. 21, V30M data shown in Fig. 18G is presented again to facilitate comparisons). Using the workflow outlined at the top of Fig. 21, plasma samples from 32 additional ATTR patients (15 WT-Cardiomyopathy, 6 V122I Cardiomyopathy, 4 T60A mixed polyneuropathy and cardiomyopathy, and 6 other mutations associated with TTR amyloidosis) were tested (see Table 1 for patient demographics & Fig. 21). A trend was observed wherein mutations associated with a primary neuropathic phenotype exhibit detectable levels of non-native oligomeric TTR (Fig. 21), whereas mutations that are associated with a primary cardiomyopathy phenotype show low or no detectable levels of oligomeric TTR (WT, VI 221). Finally, based on these experiments and results, the concentration of these oligomers is estimated to be in the low nM range within plasma (based on monomer concentration).

[00191] Unless otherwise stated, the compounds, compositions, and methods disclosed herein 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, 1 st edition, 1998).

[00192] The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all obj ectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.

[00193] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps, some will be specifically included and others specifically excluded in diverse embodiments.

[00194] Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

[00195] Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are the selection of constituent modules for the inventive compositions, and the diseases and other clinical conditions that may be diagnosed, prognosed or treated therewith. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

[00196] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[00197] In some embodiments, the terms "a," "an," and "the" and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[00198] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[00199] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[00200] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.

[00201] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described.