WO2021168156A1 - Monitoring transthyretin amyloidosis - Google Patents

Abstract

The invention provides methods of monitoring or diagnosing TTR amyloidosis from signs and/or symptoms of a subject. The invention is based in part on identification of several hitherto unrecognized signs and/or symptoms based on patient survey group and clinician survey group. Changes in signs and/or symptoms can be used, for example, to initiate or modify treatment regimens.

Description

MONITORING TRANSTHYRETIN AMYLOIDOSIS

CROSS REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit under 35 USC 119(e) of US Provisional Application No. 62/979,373, filed February 20, 2020, and of US Provisional Application No. 63/022,342, filed May 8, 2020, each of which is incorporated by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

[2] The Sequence Listing written in file 554792SEQLST.TXT is 63,188 bytes, was created on February 18, 2021, and is hereby incorporated by reference.

BACKGROUND

[3] Several diseases are thought to be caused by the abnormal folding and aggregation of disease-specific proteins. These proteins can accumulate into pathologically diagnostic accumulations, known as amyloids, which are visualized by certain histologic stains. Amyloids are thought to elicit inflammatory responses and have multiple negative consequences for the involved tissues. In addition, smaller aggregates of abnormally folded protein may exist and exert cytotoxic effects.

[4] Transthyretin (TTR) is one of the many proteins that are known to misfold and aggregate (e.g., undergo amyloidogenesis). Transthyretin-mediated amyloidosis (ATTR) encompasses two forms of disease: familial disease arising from misfolding of a mutated or variant TTR, and a sporadic, non-genetic disease caused by misfolding and aggregation of wild-type TTR. The process of TTR amyloidogenesis can cause pathology in the nervous system and/or heart, as well as in other tissues.

SUMMARY OF THE CLAIMED INVENTION

[5] The invention provides a method of controlling treatment of subjects having or at risk of a transthyretin amyloidosis comprising monitoring at least one sign and/or symptom of the subjects, wherein the at least one sign or symptom includes at least one of dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition; and initiating or modifying a treatment regimen in at least some of the subjects based on the at least one sign or symptom monitored. Optionally, the monitoring comprises determining the at least one sign or symptom of the subjects before and after initiating or modifying the treatment regimen in at least some of the subjects. Optionally, the treatment regimen comprises administering an antibody specifically binding to TTR. Optionally, the antibody reduces deposits of TTR. Optionally, the monitoring monitors at least five of the signs and/or symptoms. Optionally, the monitoring monitors all of the signs and/or symptoms.

[6] Optionally, the monitoring also monitors one or more signs and/or symptoms selected from fatigue; shortness of breath; dizziness or fainting; chest pain; sleep disturbance; pain, numbness or tingling in feet or legs; pain, numbness or tingling in hands or arms; loss of sensitivity to temperature; swelling of legs or ankles; muscle weakness or loss of strength; pain; carpal tunnel syndrome; feeling full quickly when eating; nausea; vomiting; weight loss; diarrhea; constipation; urinary incontinence; blurred vision; sexual dysfunction including erectile dysfunction, vaginal pain or dryness, decreased libido, or other sexual dysfunction; blood in urine; spinal stenosis; stress; anxiety; depression and sensitivity to alcohol. Optionally, the monitoring also monitors at least 5, 10, 15, 20, 25 or all of the signs and/or symptoms.

[7] Optionally, the method further comprises calculating an index of the subjects’ conditions from the monitored signs and/or symptoms. Optionally, the monitoring further determines a subset of the monitored signs and/or symptoms having most impact on daily life of the subjects. Optionally, the monitoring further determines severity of the monitored signs and/or symptoms. Optionally, the monitoring further determines frequency of the monitored signs and/or symptoms. Optionally, the initiating or modifying of the treatment regimen is based on the value of the index in a subject relative to one or more reference values. Optionally, the monitoring is performed weekly, monthly, quarterly, every six months or every year. Optionally, the treatment regimen is initiated when the index equals or exceeds a reference value from subjects with TTR amyloidosis. Optionally, the treatment regimen is initiated when the index differs by at least two standard deviations from the value in control subjects. Optionally, the subjects have TTR- amyloidosis and receive a treatment regimen comprising an antibody against TTR and the treatment regimen is modified based on the monitoring. Optionally, the dose or frequency of administration of the antibody is modified based on the monitoring. Optionally, the subjects have or are at risk of TTR amyloidosis, and a treatment regimen comprising an antibody against TTR is initiated based on the monitoring. Optionally, the subjects have TTR amyloidosis and are receiving a treatment regimen other than an antibody against TTR, wherein the treatment regimen is discontinued and replaced with a treatment regimen comprising an antibody against TTR based on the monitoring. Optionally, the subjects are diagnosed with TTR amyloidosis by presence of TTR deposits or a TTR level in blood. Optionally, diagnosis of TTR amyloidosis is also based on a genetic mutation in a gene encoding TTR.

[8] The invention further provides a method of treating a subject having or at risk of a transthyretin amyloidosis comprising administering an antibody specifically binding to TTR to the subject, wherein signs and/or symptoms of the subject are monitored and the signs and/or symptoms include at least one of dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition; and initiating or modifying a treatment regimen in the subject based on the symptoms.

[9] The invention further provides a method of treating a subject having or at risk of a transthyretin amyloidosis comprising administering an antibody specifically binding to TTR to the subject, and monitoring a response of the subject to the administration from signs and/or symptoms of the subject, wherein the signs and/or symptoms include at least one of dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition.

[10] The invention further provides a method of monitoring a subject having or at risk of a transthyretin amyloidosis and receiving an antibody specifically binding to TTR for treatment or prophylaxis of the amyloidosis, the method comprising monitoring signs and/or symptoms of the subject, wherein the signs and/or symptoms include at least one of dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition.

[11] The invention further provides a method of controlling treatment of a subject having or at risk of a transthyretin amyloidosis comprising monitoring signs and/or symptoms of the subject, wherein the signs and/or symptoms include at least one of dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition; and initiating or modifying a treatment regimen of the subject based on the symptoms. Optionally, the monitoring comprises determining signs and/or symptoms of the subject before and after initiating or modifying the treatment regimen. Optionally, the treatment regimen comprises administering an antibody specifically binding to TTR. Optionally, the antibody reduces deposits of TTR. Optionally, the monitoring monitors at least five of the signs and/or symptoms. Optionally, the monitoring monitors all of the signs and/or symptoms.

[12] Optionally, the monitoring also monitors one or more signs and/or symptoms selected from fatigue; shortness of breath; dizziness or fainting; chest pain; sleep disturbance; pain, numbness or tingling in feet or legs; pain, numbness or tingling in hands or arms; loss of sensitivity to temperature; swelling of legs or ankles; muscle weakness or loss of strength; pain; carpal tunnel syndrome; feeling full quickly when eating; nausea; vomiting; weight loss; diarrhea; constipation; urinary incontinence; blurred vision; sexual dysfunction including erectile dysfunction, vaginal pain or dryness, decreased libido or other sexual dysfunction; blood in urine; spinal stenosis; stress; anxiety; depression and sensitivity to alcohol. Optionally, the monitoring also monitors at least 5, 10, 15, 20, 25 or all of the signs and/or symptoms. Optionally, the method further comprises calculating an index of the subject’s conditions from the monitored signs and/or symptoms. [13] Optionally, the monitoring further determines a subset of the monitored signs and/or symptoms having most impact on daily life of the subject. Optionally, the monitoring further determines severity of the monitored signs and/or symptoms. Optionally, the monitoring further determines frequency of the monitored signs and/or symptoms. Optionally, the initiating or modifying of the treatment regimen is based on the value of the index in a subject relative to one or more reference values. Optionally, the monitoring is performed weekly, monthly, quarterly, every six months or every year. Optionally, the treatment regimen is initiated when the index equals or exceeds a reference value from subjects with TTR amyloidosis. Optionally, the treatment regimen is initiated when the index differs by at least two standard deviations from the value in control subjects. Optionally, the subject has TTR-amyloidosis and receives a treatment regimen comprising an antibody against TTR and the treatment regimen is modified based on the monitoring. Optionally, the dose or frequency of administration of the antibody is modified based on the monitoring.

[14] Optionally, the subject has or is at risk of TTR amyloidosis, and a treatment regimen comprising an antibody against TTR is initiated based on the monitoring. Optionally, wherein the subject has TTR amyloidosis and is receiving a treatment regimen other than an antibody against TTR, wherein the treatment regimen is discontinued and replaced with a treatment regimen comprising an antibody against TTR based on the monitoring. Optionally, the subject is diagnosed with TTR amyloidosis by presence of TTR deposits or a TTR level in blood. Optionally, diagnosis of TTR amyloidosis is also based on a genetic mutation in a gene encoding TTR.

[15] The invention further provides a method of diagnosing TTR amyloidosis in a subject, the method comprising: (a) monitoring signs and/or symptoms of the subject, wherein the signs and/or symptoms include at least one of dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition; and (b) diagnosing TTR amyloidosis in the subject based on the at least one sign or symptom monitored. [16] Optionally, the monitoring also monitors one or more signs and/or symptoms selected from fatigue; shortness of breath; dizziness or fainting; chest pain; sleep disturbance; pain, numbness or tingling in feet or legs; pain, numbness or tingling in hands or arms; loss of sensitivity to temperature; swelling of legs or ankles; muscle weakness or loss of strength; pain; carpal tunnel syndrome; feeling full quickly when eating; nausea; vomiting; weight loss; diarrhea; constipation; urinary incontinence; blurred vision; sexual dysfunction including erectile dysfunction, vaginal pain or dryness, decreased libido or other sexual dysfunction; blood in urine; spinal stenosis; stress; anxiety; depression and sensitivity to alcohol.

[17] Optionally, the diagnosing of TTR amyloidosis is also based on presence of TTR deposits or a TTR level in blood. Optionally, the diagnosing of TTR amyloidosis is also based on presence of a genetic mutation in a gene encoding TTR. Optionally, the diagnosing of TTR amyloidosis is also based on imaging by electrocardiogram or MRI.

BRIEF DESCRIPTION OF THE DRAWINGS

[18] Fig. 1. Most prevalent symptoms reported on worksheet: Symptoms experienced by at least 4 of 7 patients.

[19] Fig. 2. Prevalence of symptoms according to amount of burden/impact.

[20] Fig. 3. Development of ATTR-Patient Symptom Survey

[21] Fig. 4. Content included in clinician and patient cognitive debriefing interviews

[22] Fig. 5. Modifications to ATTR- Patient Symptom Survey symptom list

BRIEF DESCRIPTION OF THE SEQUENCES

[23] SEQ ID NO: 1 sets forth the amino acid sequence of a heavy chain variable region of the mouse 18C5 antibody with signal peptide.

[24] SEQ ID NO:2 sets forth a nucleic acid sequence encoding a heavy chain variable region of the mouse 18C5 antibody with signal peptide. [25] SEQ ID NO:3 sets forth the amino acid sequence of a light chain variable region of the mouse 18C5 antibody with signal peptide.

[26] SEQ ID NO:4 sets forth a nucleic acid sequence encoding a light chain variable region of the mouse 18C5 antibody with signal peptide.

[27] SEQ ID NO:5 sets forth the amino acid sequence of a Kabat/Chothia Composite CDR-H1 of the mouse 18C5 antibody.

[28] SEQ ID NO:6 sets forth a nucleic acid sequence encoding a Kabat/Chothia Composite CDR-H1 of the mouse 18C5 antibody.

[29] SEQ ID NO:7 sets forth the amino acid sequence of a Kabat/Chothia Composite CDR-H2 of the mouse 18C5 antibody.

[30] SEQ ID NO:8 sets forth a nucleic acid sequence encoding a Kabat/Chothia Composite CDR-H2 of the mouse 18C5 antibody.

[31] SEQ ID NO:9 sets forth the amino acid sequence of a Kabat/Chothia Composite CDR-H3 of the mouse 18C5 antibody.

[32] SEQ ID NO: 10 sets forth a nucleic acid sequence encoding a Kabat/Chothia Composite CDR-H3 of the mouse 18C5 antibody.

[33] SEQ ID NO: 11 sets forth the amino acid sequence of a Kabat/Chothia Composite CDR- L1 of the mouse 18C5 antibody.

[34] SEQ ID NO: 12 sets forth a nucleic acid sequence encoding a Kabat/Chothia Composite CDR-L1 of the mouse 18C5 antibody.

[35] SEQ ID NO: 13 sets forth the amino acid sequence of a Kabat/Chothia Composite CDR- L2 of the mouse 18C5 antibody.

[36] SEQ ID NO: 14 sets forth a nucleic acid sequence encoding a Kabat/Chothia Composite CDR-L2 of the mouse 18C5 antibody. [37] SEQ ID NO: 15 sets forth the amino acid sequence of a Kabat/Chothia Composite CDR- L3 of the mouse 18C5 antibody.

[38] SEQ ID NO: 16 sets forth a nucleic acid sequence encoding the a Kabat/Chothia Composite CDR-L3 of the mouse 18C5 antibody.

[39] SEQ ID NO: 17 sets forth the amino acid sequence of a chimeric 18C5 heavy chain constant region (human IgGl).

[40] SEQ ID NO: 18 sets forth a nucleic acid sequence encoding the amino acid sequence of a chimeric 18C5 heavy chain constant region (human IgGl).

[41] SEQ ID NO: 19 sets forth the amino acid sequence of a chimeric 18C5 light chain constant region (human kappa).

[42] SEQ ID NO:20 sets forth a nucleic acid sequence encoding the amino acid sequence of a chimeric 18C5 light chain constant region (human kappa).

[43] SEQ ID NO:21 sets forth the amino acid sequence of an exemplary IgGl heavy chain constant region.

[44] SEQ ID NO:22 sets forth the amino acid sequence of an exemplary IgGl Glm3 heavy chain constant region.

[45] SEQ ID NO:23 sets forth the amino acid sequence of an exemplary IgGl Glm3 heavy chain constant region.

[46] SEQ ID NO:24 sets forth the amino acid sequence of an exemplary light chain constant region with N-terminal Arginine.

[47] SEQ ID NO:25 sets forth the amino acid sequence of an exemplary light chain constant region without N-terminal Arginine.

[48] SEQ ID NO:26 sets forth the amino acid sequence of human transthyretin set forth in accession number P02766.1 (UniProt). [49] SEQ ID NO:27 sets forth the amino acid sequence of human transthyretin set forth in accession number AAB35639.1 (GenBank).

[50] SEQ ID NO:28 sets forth the amino acid sequence of human transthyretin set forth in accession number AAB35640.1 (GenBank).

[51] SEQ ID NO:29 sets forth the amino acid sequence of human transthyretin set forth in accession number and AB 163351.1 (GenBank).

[52] SEQ ID NO:30 sets forth the amino acid sequence of residues 101-109 of human transthyretin.

[53] SEQ ID NO:31 sets forth the amino acid sequence of residues 87-127 of human transthyretin.

[54] SEQ ID NO:32 sets forth a nucleic acid sequence encoding an exemplary IgGl Glm3 heavy chain constant region.

[55] SEQ ID NO:33 sets forth a nucleic acid sequence encoding an exemplary light chain constant region with C-terminal Arginine.

[56] SEQ ID NO:34 sets forth a nucleic acid sequence encoding an exemplary light chain constant region without C-terminal Arginine.

[57] SEQ ID NO:35 sets forth the amino acid sequence of a heavy chain constant region signal peptide.

[58] SEQ ID NO:36 sets forth a nucleic acid sequence encoding a heavy chain constant region signal peptide.

[59] SEQ ID NO:37 sets forth the amino acid sequence of a light chain constant region signal peptide.

[60] SEQ ID NO:38 sets forth a nucleic acid sequence encoding a light chain constant region signal peptide. [61] SEQ ID NO:39 sets forth the amino acid sequence of a Kabat CDR-H1 of antibody 14G8

[62] SEQ ID NO:40 sets forth the amino acid sequence of a Kabat CDR-H2 of antibody 14G8

[63] SEQ ID NO:41 sets forth the amino acid sequence of a Kabat CDR-H3 of antibody 14G8

[64] SEQ ID NO:42 sets forth the amino acid sequence of a Kabat CDR-L1 of antibody 14G8.

[65] SEQ ID NO:43 sets forth the amino acid sequence of a Kabat CDR-L2 of antibody 14G8.

[66] SEQ ID NO:44 sets forth the amino acid sequence of a Kabat CDR-L3 of antibody 14G8.

[67] SEQ ID NO:45 sets forth the amino acid sequence of an epitope of antibody 5A1.

[68] SEQ ID NO:46 sets forth the amino acid sequence of a Kabat CDR-H1 of antibody 5A1.

[69] SEQ ID NO:47 sets forth the amino acid sequence of a Kabat CDR-H2 of antibody 5A1.

[70] SEQ ID NO:48 sets forth the amino acid sequence of a Kabat CDR-H3 of antibody 5A1.

[71] SEQ ID NO:49 sets forth the amino acid sequence of a Kabat CDR-L1 of antibody 5A1.

[72] SEQ ID NO:50 sets forth the amino acid sequence of a Kabat CDR-L2 of antibody 5A1.

[73] SEQ ID NO:51 sets forth the amino acid sequence of a Kabat CDR-L3 of antibody 5A1.

[74] SEQ ID NO:52 sets forth the amino acid sequence of a Kabat CDR-H1 of antibody 6C1.

[75] SEQ ID NO:53 sets forth the amino acid sequence of a Kabat CDR-H2 of antibody 6C1.

[76] SEQ ID NO:54 sets forth the amino acid sequence of a Kabat CDR-H3 of antibody 6C1.

[77] SEQ ID NO: 55 sets forth the amino acid sequence of a Kabat CDR-L1 of antibody 6C1.

[78] SEQ ID NO:56 sets forth the amino acid sequence of a Kabat CDR-L2 of antibody 6C1.

[79] SEQ ID NO:57 sets forth the amino acid sequence of a Kabat CDR-L3 of antibody 6C1.

[80] SEQ ID NO:58 sets forth the amino acid sequence of a VH region of antibody AD7F6. [81] SEQ ID NO:59 sets forth the amino acid sequence of a VL region of antibody AD7F6.

[82] SEQ ID NO:60 sets forth the amino acid sequence of a CDR-H1 of antibody RT24.

[83] SEQ ID NO:61 sets forth the amino acid sequence of a CDR-H2 of antibody RT24.

[84] SEQ ID NO:62 sets forth the amino acid sequence of a CDR-H3 of antibody RT24.

[85] EQ ID NO:63 sets forth the amino acid sequence of a CDR-L1 of antibody RT24.

[86] SEQ ID NO:64 sets forth the amino acid sequence of a CDR-L2 of antibody RT24.

[87] SEQ ID NO:65 sets forth the amino acid sequence of a CDR-L3 of antibody RT24.

[88] SEQ ID NO:66 sets forth the amino acid sequence of a CDR-H1 of antibody NI-

301.35G11.

[89] SEQ ID NO: 67 sets forth the amino acid sequence of a CDR-H2 of antibody NI- 301.35G11.

[90] SEQ ID NO: 68 sets forth the amino acid sequence of a CDR-H3 of antibody NI- 301.35G11.

[91] SEQ ID NO:69 sets forth the amino acid sequence of a CDR-L1 of antibody NI- 301.35G11.

[92] SEQ ID NO:70 sets forth the amino acid sequence of a CDR-L2 of antibody NI- 301.35G11.

[93] SEQ ID NO:71 sets forth the amino acid sequence of a CDR-L3 of antibody NI- 301.35G11.

[94] SEQ ID NO: 72 sets forth the amino acid sequence of an epitope of antibodies MFD101, MDF102, MFD103, MFD105.

[95] SEQ ID NO: 73 sets forth the amino acid sequence of an epitope of antibodies MFD 107, MFD108, MFD 109, MFD111. [96] SEQ ID NO:74 sets forth the amino acid sequence of an epitope of antibody MFD114.

[97] SEQ ID NO:75 sets forth the amino acid sequence of a Kabat CDR-H1 of antibody 9D5.

[98] SEQ ID NO:76 sets forth the amino acid sequence of a Kabat CDR-H2 of antibody 9D5.

[99] SEQ ID NO:77 sets forth the amino acid sequence of a Kabat CDR-H3 of antibody 9D5.

[100] SEQ ID NO:78 sets forth the amino acid sequence of a Kabat CDR-L1 of antibody 9D5.

[101] SEQ ID NO:79 sets forth the amino acid sequence of a Kabat CDR-L2 of antibody 9D5.

[102] SEQ ID NO:80 sets forth the amino acid sequence of a Kabat CDR-L3 of antibody 9D5.

[103] SEQ ID NO:81 sets forth the amino acid sequence of a mature heavy chain variable region of the mouse 18C5 antibody.

[104] SEQ ID NO:82 sets forth the amino acid sequence of a heavy chain variable region of the murine anti-pyroglutamate-Abeta antibody Fab c#17, GenBank Acc. No. 1212215935.

[105] SEQ ID NO:83 sets forth the amino acid sequence of a heavy chain variable region of humanized Crenezumab Fab (CreneFab) PDB: 5VZY, GenBank Acc. No. 1229749875.

[106] SEQ ID NO:84 sets forth the amino acid sequence of a heavy chain variable region of the human germline sequence IGHV3-48*01, GenBank Acc. No. 1FN550289.1.

[107] SEQ ID NO:85 sets forth the amino acid sequence of a heavy chain variable region of the humanized 18C5 antibody hul8C5-VH_l.

[108] SEQ ID NO:86 sets forth the amino acid sequence of a heavy chain variable region of the humanized 18C5 antibody hul8C5-VH_2.

[109] SEQ ID NO:87 sets forth the amino acid sequence of a mature light chain variable region of the mouse 18C5 antibody.

[110] SEQ ID NO:88 sets forth the amino acid sequence of a light chain variable region of the murine anti-pyroglutamate-Abeta antibody Fab c#17, GenBank Acc. No. 1212215934. [111] SEQ ID NO:89 sets forth the amino acid sequence of a light chain variable region of humanized Crenezumab Fab (CreneFab) PDB: 5VZY, GenBank Acc. No. 1229749876.

[112] SEQ ID NO:90 sets forth the amino acid sequence of a light chain variable region of the human germline sequence IGKV2-30*2, GenBank Acc. No. CAA77315.

[113] SEQ ID NO:91 sets forth the amino acid sequence of a light chain variable region of the humanized 18C5 antibody hul8C5-VL_l.

[114] SEQ ID NO:92 sets forth the amino acid sequence of a light chain variable region of the humanized 18C5 antibody hul8C5-VL_2.

[115] SEQ ID NO:93 sets forth the amino acid sequence of Kabat CDR-H1 of the mouse 18C5 antibody.

[116] SEQ ID NO:94 sets forth the amino acid sequence of Chothia CDR-H1 of the mouse 18C5 antibody.

[117] SEQ ID NO:95 sets forth the amino acid sequence of Contact CDR-H1 of the mouse 18C5 antibody.

[118] SEQ ID NO:96 sets forth the amino acid sequence of Chothia CDR-H2 of the mouse 18C5 antibody.

[119] SEQ ID NO:97 sets forth the amino acid sequence of AbM CDR-H2 of the mouse 18C5 antibody.

[120] SEQ ID NO: 98 sets forth the amino acid sequence of Contact CDR-H2 of the mouse 18C5 antibody.

[121] SEQ ID NO:99 sets forth the amino acid sequence of Contact CDR-H3 of the mouse 18C5 antibody.

[122] SEQ ID NO: 100 sets forth the amino acid sequence of Contact CDR-L1 of the mouse 18C5 antibody. [123] SEQ ID NO: 101 sets forth the amino acid sequence of Contact CDR-L2 of the mouse 18C5 antibody.

[124] SEQ ID NO: 102 sets forth the amino acid sequence of Contact CDR-L3 of the mouse 18C5 antibody.

[125] SEQ ID NO: 103 sets forth the amino acid sequence of a heavy chain variable region of the mouse 9D5 antibody.

[126] SEQ ID NO: 104 sets forth the amino acid sequence of a light chain variable region of the mouse 9D5 antibody.

DEFINITIONS

[127] Monoclonal antibodies or other biological entities are typically provided in isolated form. This means that an antibody or other biologically entity is typically at least 50% w/w pure of interfering proteins and other contaminants arising from its production or purification but does not exclude the possibility that the monoclonal antibody is combined with an excess of pharmaceutically acceptable carrier(s) or other vehicle intended to facilitate its use. Sometimes monoclonal antibodies are at least 60%, 70%, 80%, 90%, 95% or 99% w/w pure of interfering proteins and contaminants from production or purification. Often an isolated monoclonal antibody or other biological entity is the predominant macromolecular species remaining after its purification.

[128] Specific binding of an antibody to its target antigen means an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M ¹. Specific binding is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit ( e.g ., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Specific binding does not however necessarily imply that an antibody binds one and only one target.

[129] The basic antibody structural unit is a tetramer of subunits. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region means a light chain variable region without the light chain signal peptide. The carboxy -terminal portion of each chain defines a constant region primarily responsible for effector function.

[130] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. See generally, Fundamental Immunology , Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989, Ch. 7 (incorporated by reference in its entirety for all purposes).

[131] An immunoglobulin light or heavy chain variable region (also referred to herein as a “light chain variable domain” (“VL domain”) or “heavy chain variable domain” (“VH domain”), respectively) consists of a “framework” region interrupted by three “complementarity determining regions” or “CDRs.” The framework regions serve to align the CDRs for specific binding to an epitope of an antigen. The CDRs include the amino acid residues of an antibody that are primarily responsible for antigen binding. From amino-terminus to carboxyl-terminus, both VL and VH domains comprise the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of a VL domain are also referred to herein, respectively, as CDR-L1, CDR-L2, and CDR-L3; CDRs 1, 2, and 3 of a VH domain are also referred to herein, respectively, as CDR-H1, CDR-H2, and CDR-H3.

[132] The assignment of amino acids to each VL and VH domain is in accordance with any conventional definition of CDRs. Conventional definitions include, the Rabat definition (Rabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), The Chothia definition (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et ah, Nature 342:878-883, 1989); a composite of Chothia Rabat CDR in which CDR- H1 is a composite of Chothia and Rabat CDRs; the AbM definition used by Oxford Molecular’s antibody modelling software; and, the contact definition of Martin et al (bioinfo.org.uk/abs) (see Table 1). Kabat provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. When an antibody is said to comprise CDRs by a certain definition of CDRs (e.g., Kabat) that definition specifies the minimum number of CDR residues present in the antibody (i.e., the Kabat CDRs). It does not exclude that other residues falling within another conventional CDR definition but outside the specified definition are also present. For example, an antibody comprising CDRs defined by Kabat includes among other possibilities, an antibody in which the CDRs contain Kabat CDR residues and no other CDR residues, and an antibody in which CDR HI is a composite Chothia-Kabat CDR HI and other CDRs contain Kabat CDR residues and no additional CDR residues based on other definitions.

Table 1

Conventional Definitions of CDRs Using Kabat Numbering

*CDR-H1 by Chothia can end at H32, H33, or H34 (depending on the length of the loop). This is because the Kabat numbering scheme places insertions of extra residues at 35A and 35B, whereas Chothia numbering places them at 31 A and 3 IB. If neither H35A nor H35B (Kabat numbering) is present, the Chothia CDR- H1 loop ends at H32. If only H35A is present, it ends at H33. If both H35A and H35B are present, it ends at H34. [133] The term “antibody” includes intact antibodies and binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to the target including separate heavy chains, light chains Fab, Fab', F(ab')2, F(ab)c, Dabs, nanobodies, and Fv. Fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins. The term “antibody” also includes a bispecific antibody and/or a humanized antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy /light chain pairs and two different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol ., 79:315-321 (1990); Kostelny et ah, J. Immunol ., 148:1547-53 (1992)). In some bispecific antibodies, the two different heavy /light chain pairs include a humanized 9D5 heavy chain/light chain pair and a heavy chain/light chain pair specific for a different epitope on transthyretin than that bound by 9D5. In some bispecific antibodies, the two different heavy /light chain pairs include a humanized 18C5 heavy chain/light chain pair and a heavy chain/light chain pair specific for a different epitope on transthyretin than that bound by 18C5.

[134] In some bispecific antibodies, one heavy chain/light chain pair is a humanized 9D5 antibody or a humanized 18C5 antibody as further disclosed below and the other heavy chain/light chain pair is from an antibody that binds to a receptor expressed on the blood brain barrier, such as an insulin receptor, an insulin-like growth factor (IGF) receptor, a leptin receptor, or a lipoprotein receptor, or a transferrin receptor (Friden et ah, Proc. Natl. Acad. Sci. USA 88:4771-4775, 1991; Friden et al, Science 259:373-377, 1993). Such abispecific antibody can be transferred cross the blood brain barrier by receptor-mediated transcytosis. Brain uptake of the bispecific antibody can be further enhanced by engineering the bispecific antibody to reduce its affinity to the blood brain barrier receptor. Reduced affinity for the receptor resulted in a broader distribution in the brain (see, e.g, Atwal et ah, Sci. Trans. Med. 3, 84ra43, 2011; Yu et al, Sci. Trans. Med. 3, 84ra44, 2011).

[135] Exemplary bispecific antibodies can also be: (1) a dual-variable-domain antibody (DVD- Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu etal. , Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (3) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (4) a so- called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; or (5) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fc-region. Examples of platforms useful for preparing bispecific antibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgGl (Xencor) or DuoBody (based on Fab arm exchange, Genmab).

[136] The term “epitope” refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). The epitope can be linear, such as an epitope of, for example, 2-5, 3-5, 3-9, or 5-9 contiguous amino acids from SEQ ID NO:26, including for example, two or more contiguous amino acids within residues 89-97 of the mature region of SEQ ID NO:26. The epitope can also be a conformational epitope including, for example, two or more non-contiguous segments of amino acids within residues 89-97 of the mature region of SEQ ID NO:26. If an antibody is said to bind to an epitope within amino acid residues 89-97 of transthyretin (TTR) (the mature region of SEQ ID NO:26), for example, what is meant is that the epitope is within the recited range of amino acids including those defining the outer-limits of the range. It does not necessarily mean that every amino acid within the range constitutes part of the epitope. Thus, for example, an epitope within amino acid residues 89-97 of TTR may consist of amino acids 89-97, 89-96, 90-97, 89-95, 90-96, 91-97, 89-94, 90-95, 91-96, 92-97, 89-93, 90-94, 91-95, 92-96, 93-97, 89-92, 90-93, 91-94, 92-95, 93-96, 94-97, 89-91, 90-92, 91-93, 92-94, 93- 95, 94-96, 95-97 of SEQ ID NO:26, among other linear segments of SEQ ID NO:45, or in the case of conformational epitopes, non-contiguous segments of amino acids of SEQ ID NO:45. Epitopes can include E89K and E89Q substitutions, E being the wildtype residue).

[137] The epitope can be linear, such as an epitope of, for example, 2-5, 3-5, 3-9, or 5-9 contiguous amino acids from SEQ ID NO:26, including for example, two or more contiguous amino acids within residues 101-109 of the mature region of SEQ ID NO:26. The epitope can also be a conformational epitope including, for example, two or more non-contiguous segments of amino acids within residues 101-109 of the mature region of SEQ ID NO:26. If an antibody is said to bind to an epitope within amino acid residues 101-109 of transthyretin (TTR) (the mature region of SEQ ID NO:26), for example, what is meant is that the epitope is within the recited range of amino acids including those defining the outer-limits of the range. It does not necessarily mean that every amino acid within the range constitutes part of the epitope. Thus, for example, an epitope within amino acid residues 101-109 of TTR may consist of amino acids 101-109, 101-108, 102-109, 101-107, 102-108, 103-109, 101-106, 102-107, 103-108, 104-109,

101-105, 102-106, 103-107, 104-108, 105-109, 101-104, 102-105, 103-106, 104-107, 105-108,

106-109, 101-103, 102-104, 103-105, 104-106, 105-107, 106-108, 107-109, 101-102, 102-103,

103-104, 104-105, 105-106, 106-107, 107-108, or 108-109 of SEQ ID NO:26, among other linear segments of SEQ ID NO: 30, or in the case of conformational epitopes, non-contiguous segments of amino acids of SEQ ID NO:30.

[138] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen. The epitope of an antibody can also be defined by X-ray crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[139] Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al, Cancer Res. 50: 1495, 1990). A test antibody competes with a reference antibody if an excess of a test antibody ( e.g ., at least 2x, 5x, lOx, 20x or lOOx) inhibits binding of the reference antibody by at least 50% as measured in a competitive binding assay. Some test antibodies inhibit binding of the references antibody by at least 75%, 90% or 99%. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.

[140] The term “native” with respect to the structure transthyretin (TTR) refers to the normal folded structure of TTR in its properly functioning state (i.e., a TTR tetramer). As TTR is a tetramer in its natively folded form, non-native forms of TTR include, for example, misfolded TTR tetramers, TTR monomers, aggregated forms of TTR, and fibril forms of TTR. Non-native forms of TTR can include molecules comprising wild-type TTR amino acid sequences or mutations.

[141] The term “misfolded” with respect to TTR refers to the secondary and tertiary structure of a TTR polypeptide monomer or multimer, and indicates that the polypeptide has adopted a conformation that is not normal for that protein in its properly functioning state. Although TTR misfolding can be caused by mutations in the protein (e.g., deletion, substitution, or addition), wild-type TTR proteins can also be misfolded in diseases, exposing specific epitopes.

[142] The term “pharmaceutically acceptable” means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

[143] The terms “subject” and “patient” are used interchangeably to include human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[144] An individual is at risk of a disease if the subject has at least one known risk-factor (e.g., genetic, biochemical, family history, and situational exposure) placing individuals with that risk factor at a statistically significant greater risk of developing the disease than individuals without the risk factor.

[145] The term “biological sample” refers to a sample of biological material within or obtainable from a biological source, for example a human or mammalian subject. Such samples can be organs, organelles, tissues, sections of tissues, bodily fluids, peripheral blood, blood plasma, blood serum, cells, molecules such as proteins and peptides, and any parts or combinations derived therefrom. The term biological sample can also encompass any material derived by processing the sample. Derived material can include cells or their progeny.

Processing of the biological sample may involve one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, and the like.

[146] The term “control sample” refers to a biological sample not known or suspected to include monomeric, misfolded, aggregated, or fibril forms of transthyretin (TTR), such as in TTR amyloid deposits. Control samples can be obtained from individuals not afflicted with a TTR amyloidosis or a specifically chosen type of TTR amyloidosis. Alternatively, control samples can be obtained from subjects afflicted with TTR amyloidosis or a specifically chosen type of TTR amyloidosis. Such samples can be obtained at the same time as a biological sample thought to comprise the TTR amyloidosis or on a different occasion. A biological sample and a control sample can both be obtained from the same tissue (e.g., a tissue section containing both TTR amyloid deposits and surrounding normal tissue). Preferably, control samples consist essentially or entirely of tissue free of TTR amyloid deposits and can be used in comparison to a biological sample thought to comprise TTR amyloid deposits. Preferably, the tissue in the control sample is the same type as the tissue in the biological sample (e.g., cardiomyocytes in the heart).

[147] The term “disease” refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology.

[148] The term “symptom” refers to a subjective evidence of a disease, such as altered gait, as perceivable by a subject. A "sign" refers to objective evidence of a disease as observable by a physician. Reference to monitoring sign(s) and/or symptoms(s) of a subject can be accomplished by monitoring a sign, signs, symptom, symptoms, a sign and a symptom, signs and symptoms, a sign and symptoms, signs and a symptom or any other combination. Practice of the invention does not necessarily require definitive classification of whether a subject characteristic is a sign or symptom.

[149] For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): asn, gin, cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another.

[150] Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject antibody region ( e.g ., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.

[151] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” an antibody may contain the antibody alone or in combination with other ingredients.

[152] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.

[153] Unless otherwise apparent from the context, the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value.

[154] Statistical significance means p<0.05.

[155] Antibodies of the invention can be administered concomitant with another treatment for the same indication as the antibody, meaning that the other treatment is administered at least once during the period in which the antibody is administered, such period beginning one month before the first dosing and ending one month after the last dosing of the antibody. The other treatment can be administered at recurring intervals during this period, which may or may not be the same as the intervals at which the antibody is administered. The other treatment may be a symptomatic treatment.

[156] A treatment is symptomatic if it only affects one or more symptoms of a disease, not its cause, i.e., its etiology.

[157] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” can include a plurality of compounds, including mixtures thereof.

DETAILED DESCRIPTION

I. General

[158] The invention provides methods of monitoring TTR amyloidosis from signs and/or symptoms of a subject. The invention is based in part on identification of several hitherto unrecognized signs and/or symptoms based on patient survey group. Changes in signs and/or symptoms can be used, for example, to initiate or modify treatment regimens, or to diagnose TTR amyloidosis.

II. Target Molecules

[159] Transthyretin (TTR) is a 127-amino acid, 55 kDa serum and cerebrospinal fluid transport protein primarily synthesized by the liver. It has also been referred to as prealbumin, thyroxine binding prealbumin, ATTR, and TBPA. In its native state, TTR exists as a tetramer. In homozygotes, the tetramers comprise identical 127-amino-acid beta-sheet-rich subunits. In heterozygotes, the TTR tetramers are made up of variant and/or wild-type subunits, typically combined in a statistical fashion.

[160] The established function of TTR in the blood is to transport holo-retinol binding protein. Although TTR is the major carrier of thyroxine (T4) in the blood of rodents, utilizing binding sites that are orthogonal to those used for holo-retinol binding protein, the T4 binding sites are effectively unoccupied in humans. [161] TTR is one of at least thirty different human proteins whose extracellular misfolding and/or misassembly (amyloidogenesis) into a spectrum of aggregate structures is thought to cause degenerative diseases referred to as amyloid diseases. TTR undergoes conformational changes in order to become amyloidogenic. Dissociation of the TTR tetramer and partial unfolding exposes stretches of largely uncharged hydrophobic residues in an extended conformation that efficiently misassemble into largely unstructured spherical aggregates that ultimately undergo conformation conversion into cross-beta sheet amyloid structures.

[162] Unless otherwise apparent from context, reference to transthyretin (TTR) or its fragments or domains includes the natural human amino acid sequences including isoforms, mutants (e.g., E89K and E89Q), and allelic variants thereof. Exemplary TTR polypeptide sequences are designated by Accession Numbers P02766.1 (UniProt) (SEQ ID NO:26), AAB35639.1 (GenBank) (SEQ ID NO:27), AAB35640.1 (GenBank) (SEQ ID NO:28), and ABI63351.1 (GenBank) (SEQ ID NO:29). Residues are numbered according to Swiss Prot P02766.1, with the first amino acid of the mature protein ( i.e ., not including the 20 amino acid signal sequence) designated residue 1. In any other TTR protein, residues are numbered according to the corresponding residues in P02766.1 on maximum alignment.

III. Transthyretin Amyloidosis

[163] Transthyretin (TTR) amyloidosis is a systemic disorder characterized by pathogenic, misfolded TTR and the extracellular deposition of amyloid fibrils composed of TTR. TTR amyloidosis is generally caused by destabilization of the native TTR tetramer form (due to environmental or genetic conditions), leading to dissociation, misfolding, and aggregation of TTR into amyloid fibrils that accumulate in various organs and tissues, causing progressive dysfunction. See, e.g., Almeida and Saraiva, FEBS Letters 586:2891-2896 (2012); Ando et al, Orphanet Journal of Rare Diseases 8:31 (2013).

[164] In humans, both wild-type TTR tetramers and mixed tetramers comprised of mutant and wild-type subunits can dissociate, misfold, and aggregate, with the process of amyloidogenesis leading to the degeneration of affected tissue. Thus, TTR amyloidoses encompass diseases caused by pathogenic misfolded TTR resulting from mutations in TTR or resulting from non- mutated, misfolded TTR. [165] For example, wild-type ATTR amyloidosis (also called senile systemic amyloidosis or SSA) and senile cardiac amyloidosis (SCA) are age-related types of amyloidosis that result from the deposition of wild-type TTR amyloid outside and within the cardiomyocytes of the heart. TTR amyloidosis is also the most common form of hereditary (familial) amyloidosis, which is caused by mutations that destabilize the TTR protein. The TTR amyloidoses associated with point mutations in the TTR gene include familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), and the rare central nervous system selective amyloidosis (CNSA). Patients with hereditary (familial) TTR amyloidosis are almost always heterozygotes, meaning that the TTR tetramers are composed of mutant and/or wild-type TTR subunits, generally statistically distributed. Hereditary (familial) versions of TTR amyloidosis are generally autosomal dominant and are typically earlier onset than the sporadic diseases (SSA and SCA).

[166] There are over 100 mutations in the gene encoding TTR that have been implicated in the autosomal dominant disorders FAP and FAC. See, e.g., US 2014/0056904; Saraiva, Hum.

Mutat. 17(6):493-503 (2001); Damas and Saraiva, J Struct. Biol. 130:290-299; Dwulet and Benson, Biochem. Biophys. Res. Commun. 114:657-662 (1983). These amyloid-causing mutations are distributed throughout the entire molecule of TTR. Generally, the more destabilizing the mutant subunits are to the TTR tetramer structure, the earlier the onset of amyloid disease. The pathogenic potential of a TTR variant is generally determined by a combination of its instability and its cellular secretion efficiency. The initial pathology caused by some TTR variants comes from their selective destruction of cardiac tissue, whereas that from other TTR variants comes from compromising the peripheral and autonomic nervous system.

The tissue damage caused by TTR amyloidogenesis appear to stem largely from the toxicity of small, diffusible TTR aggregates, although accumulation of extracellular amyloid may contribute and almost certainly compromises organ structure in the late stages of the TTR amyloidosis. Exemplary TTR mutations include V30M, Y114C, G47R, S50I, E61L, T49S, F33V, A45T, E89K, E89Q, and V122I.

[167] TTR amyloidosis presents in many different forms, with considerable phenotypic variation across individuals and geographic locations. For example, TTR amyloidosis can present as a progressive, axonal sensory autonomic and motor neuropathy. TTR amyloidosis can also present as an infiltrative cardiomyopathy.

[168] The age at onset of disease-related signs and/or symptoms varies between the second and ninth decades of life, with great variations across different populations. The multisystem involvement of TTR amyloidosis is a clue to its diagnosis. For example, TTR amyloidosis diagnosis is considered when one or several of the following are present: (1) family history of neuropathic disease, especially associated with heart failure; (2) neuropathic pain or progressive sensory disturbances of unknown etiology; (3) carpal tunnel syndrome without obvious cause, particularly if it is bilateral and requires surgical release; (4) gastrointestinal motility disturbances or autonomic nerve dysfunction of unknown etiology ( e.g ., erectile dysfunction, orthostatic hypotension, neurogenic bladder); (5) cardiac disease characterized by thickened ventricular walls in the absence of hypertension; (6) advanced atrio- ventricular block of unknown origin, particularly when accompanied by a thickened heart; and (6) vitreous body inclusions of the cotton-wool type. See Ando et al, Orphanet Journal of Rare Diseases 8:31 (2013). Other signs and/or symptoms can include, for example, polyneuropathy, sensory loss, pain, weakness in lower limbs, dyshidrosis, diarrhea, constipation, weight loss, and urinary incontinence/ retenti on .

[169] Diagnosis of TTR amyloidosis typically relies on target organ biopsies, followed by histological staining of the excised tissue with the amyloid-specific dye, Congo red. If a positive test for amyloid is observed, immunohistochemical staining and mass spectroscopic identification of TTR is subsequently performed to ensure that the precursor protein responsible for amyloid formation is indeed TTR. Antibodies disclosed herein are useful in distinguishing TTR amyloidosis from a non-TTR amyloidosis e.g. amyloid light-chain (AL) amyloidosis, also known as primary systemic amyloidosis. For familial forms of the diseases, demonstration of a mutation in the gene encoding TTR is then needed before diagnosis can be made. This can be accomplished, for example, through isoelectric focusing electrophoresis, polymerase chain reaction, or laser dissection/liquid chromatography-tandem mass spectrometry. See, e.g., US 2014/0056904; Ruberg and Berk, Circulation 126:1286-1300 (2012); Ando et al., Orphanet Journal of Rare Diseases 8:31 (2013). IV. Therapies for TTR amyloidosis

[170] In some methods of the invention the therapy or treatment for treating TTR amyloidosis is selected from the group comprising small molecules, antisense oligonucleotides, small interfering RNAs, and antibodies. In some embodiments, the TTR amyloidosis therapy stabilizes TTR, including TTR tetramers. In some embodiments, the TTR amyloidosis therapy inhibits TTR protein synthesis. In some embodiments, the TTR amyloidosis therapy degrades mutant and wild-type TTR mRNA through RNA interference. In some embodiments, the TTR amyloidosis therapy reduces serum TTR protein and TTR deposits in tissue. In some embodiments, the TTR amyloidosis therapy disrupts TTR fibril formation. In some embodiments, the TTR amyloidosis therapy reduces nonfibrillar TTR deposition. In some embodiments, the TTR amyloidosis therapy reduces monomeric, misfolded, aggregated, or fibril forms of TTR.

[171] In some embodiments, the TTR amyloidosis treatment is VYNDAQEL® (tafamidis meglumine), VYNDAMAX™ (tafamidis), ONPATTRO™ (patisiran), TEGSEDI™ (inotersen), diflunisal, doxycycline, tauroursodeoxycholic, tolcapone, an anti-serum amyloid P agent, or an anti-TTR antibody, including those described herein.

V. Antibodies

A. Binding Specificity and Functional Properties

[172] Some methods of the invention use monoclonal antibodies binding to transthyretin (TTR) protein, for example, to epitopes within amino acid residues 89-97 (SEQ ID NO:45) or to epitopes within amino acid residues 101-109 (SEQ ID NO: 30) of TTR. Such epitopes are buried in the native TTR tetramer and exposed in monomeric, misfolded, aggregated, or fibril forms of TTR.

[173] Such antibodies include 9D5, 18C5, and their chimeric, veneered and humanized forms. 9D5 specifically binds within amino acid residues 89-97 (SEQ ID NO:45) of TTR. 18C5 specifically binds within amino acid residues 101-109 (SEQ ID NO: 30) of TTR. These antibodies are further characterized by their ability to bind to monomeric, misfolded, aggregated, or fibril forms of TTR but not to native tetrameric form of TTR. Ability to bind to specific proteins or fragments thereof may be demonstrated using exemplary assay formats provided in the examples. Unless otherwise apparent from the context, reference to 9D5 or 18C5 should be understood as referring to any of the mouse, chimeric, veneered or humanized forms. A hybridoma cell line that produces monoclonal antibody 9D5 was deposited with the Patent Depository of the American Type Culture Collection (ATCC), Manassas, Virginia, 20110-2209 on April 4, 2017 and assigned Patent Deposit No. PTA-124078. A hybridoma cell line that produces monoclonal antibody 18C5 was deposited with the Patent Depository of the American Type Culture Collection (ATCC), Manassas, Virginia, 20110-2209 on October 31, 2017 and assigned Patent Deposit No. PTA-124570.

[174] Some antibodies bind to the same or overlapping epitope as an antibody designated 9D5. The sequences of the heavy and light chain mature variable regions of 9D5 are designated SEQ ID NOs: 81 and 82, respectively. Other antibodies having such a binding specificity can be produced by immunizing mice with TTR, or a portion thereof including the desired epitope (e.g., SEQ ID NO:45), and screening resulting antibodies for binding to monomeric TTR or a peptide comprising SEQ ID NO:45, optionally in competition with an antibody having the variable regions of mouse 18C5 (IgGl, kappa). Fragments of TTR including the desired epitope can be linked to a carrier that helps elicit an antibody response to the fragment and/or be combined with an adjuvant that helps elicit such a response. Such antibodies can be screened for differential binding to wild-type, monomeric versions of TTR or a fragment thereof (e.g., SEQ ID NO:26) compared with mutants of specified residues.

[175] Some antibodies bind to the same or overlapping epitope as an antibody designated 18C5. The sequences of the heavy and light chain mature variable regions of 18C5 are designated SEQ ID NOs: 1 and 3, respectively. Other antibodies having such a binding specificity can be produced by immunizing mice with TTR, or a portion thereof including the desired epitope (e.g., SEQ ID NO: 30), and screening resulting antibodies for binding to monomeric TTR or a peptide comprising SEQ ID NO:30, optionally in competition with an antibody having the variable regions of mouse 18C5 (IgGl, kappa). Fragments of TTR including the desired epitope can be linked to a carrier that helps elicit an antibody response to the fragment and/or be combined with an adjuvant that helps elicit such a response. Such antibodies can be screened for differential binding to wild-type, monomeric versions of TTR or a fragment thereof (e.g, SEQ ID NO:26) compared with mutants of specified residues. [176] Screening against such mutants more precisely defines the binding specificity to allow identification of antibodies whose binding is inhibited by mutagenesis of particular residues and which are likely to share the functional properties of other exemplified antibodies. The mutations can be systematic replacement substitution with alanine (or serine or glycine if an alanine is present already) one residue at a time, or more broadly spaced intervals, throughout the target or throughout a section thereof in which an epitope is known to reside. If the same set of mutations significantly reduces the binding of two antibodies, the two antibodies bind the same epitope.

[177] Antibodies having the binding specificity of a selected murine antibody ( e.g ., 9D5 or 18C5) can also be produced using a variant of the phage display method. See Winter, WO 92/20791. This method is particularly suitable for producing human antibodies. In this method, either the heavy or light chain variable region of the selected murine antibody is used as a starting material. If, for example, a light chain variable region is selected as the starting material, a phage library is constructed in which members display the same light chain variable region

( i.e ., the murine starting material) and a different heavy chain variable region. The heavy chain variable regions can for example be obtained from a library of rearranged human heavy chain variable regions. A phage showing strong specific binding (e.g., at least 10⁸ and preferably at least 10⁹ M ¹) for monomeric TTR or a fragment thereof (e.g, amino acid residues 89-97 or amino acid residues 101-109) is selected. The heavy chain variable region from this phage then serves as a starting material for constructing a further phage library. In this library, each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region. The light chain variable regions can be obtained for example from a library of rearranged human variable light chain regions. Again, phage showing strong specific binding for monomeric TTR or a fragment thereof (e.g, amino acid residues 89-97 or amino acid residues 101-109) are selected. The resulting antibodies usually have the same or similar epitope specificity as the murine starting material.

[178] Other antibodies can be obtained by mutagenesis of cDNA encoding the heavy and light chains of an exemplary antibody, such as 9D5 or 18C5. Monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to 9D5 or 18C5 in amino acid sequence of the mature heavy and/or light chain variable regions and maintain its functional properties, and/or which differ from the respective antibody by a small number of functionally inconsequential amino acid substitutions ( e.g ., conservative substitutions), deletions, or insertions are also included in the invention. Monoclonal antibodies having at least one or all six CDR(s) as defined by conventional definition, but preferably Kabat, that are 90%, 95%, 99% or 100% identical to corresponding CDRs of 9D5 or 18C5 are also included.

[179] Some methods of the invention also use antibodies having some or all (e.g., 3, 4, 5, and 6) CDRs entirely or substantially from 9D5 or 18C5. Such antibodies can include a heavy chain variable region that has at least two, and usually all three, CDRs entirely or substantially from the heavy chain variable region of 9D5 or 18C5 and/or a light chain variable region having at least two, and usually all three, CDRs entirely or substantially from the light chain variable region of 9D5 or 18C5. The antibodies can include both heavy and light chains. A CDR is substantially from a corresponding 9D5 or 18C5 CDR when it contains no more than 4, 3, 2, or 1 substitutions, insertions, or deletions, except that CDR-H2 (when defined by Kabat) can have no more than 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions. Such antibodies can have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to 9D5 or 18C5 in the amino acid sequence of the mature heavy and/or light chain variable regions and maintain their functional properties, and/or differ from 9D5 or 18C5 by a small number of functionally inconsequential amino acid substitutions (e.g, conservative substitutions), deletions, or insertions.

[180] Kabat CDRs (CDR-H1, CDR-H2, CDR-H3) of the heavy chain of 9D5 are designated SEQ ID NOs: 75, 76, and 77, respectively, and Kabat CDRs (CDR-L1, CDR-L2, CDR-L3) of the light chain of 9D5 are designated SEQ ID NOs: 78, 79, and 80, respectively.

[181] Kabat/Chothia Composite CDRs (CDR-H1, CDR-H2, CDR-H3) of the heavy chain of 18C5 are designated SEQ ID NOs: 5, 7, and 9, respectively, and Kabat/Chothia Composite CDRs (CDR-L1, CDR-L2, CDR-L3) of the light chain of 18C5 are designated SEQ ID NOs: 11, 13, and 15, respectively.

[182] Table 2 indicates the 18C5 CDRs as defined by Kabat, Chothia, Composite of Chothia and Kabat (also referred to herein as “Kabat/Chothia Composite”), AbM, and Contact. Table 2

18C5 CDRs as defined by Kabat, Chothia, Composite of Chothia and Kabat, AbM, and

Contact, Using Kabat Numbering

[183] Some antibodies identified by such assays can bind to monomeric, misfolded, aggregated, or fibril forms of TTR but not to native tetrameric form of TTR, as described in the examples or otherwise. Likewise, some antibodies are immunoreactive on TTR-mediated amyloidosis tissue but not on healthy tissue.

[184] Some antibodies can inhibit or reduce aggregation of TTR, inhibit or reduce TTR fibril formation, reduce or clear TTR deposits or aggregated TTR, or stabilize non-toxic conformations of TTR in an animal model or clinical trial. Some antibodies can treat, effect prophylaxis of, or delay the onset of a TTR amyloidosis as shown in an animal model or clinical trial. Exemplary animal models for testing activity against a TTR amyloidosis include those described in Kohno et al., Am. J. Path. 150(4):1497-1508 (1997); Teng et al, Laboratory Investigations 81:385-396 (2001); Wakasugi etal, Proc. Japan Acad. 63B:344-347 (1987); Shimada etal, Mol. Biol. Med. 6:333-343 (1989); Nagata et al, J. Biochem. 117:169-175 (1995); Sousa et al, Am. J. Path. 161:1935-1948 (2002); and Santos etal., Neurobiology of Aging 31:280-289 (2010).

[185] Anti-TTR antibodies including chimeric and humanized versions thereof, are useful in combination therapies, in bispecific antibodies, in methods of diagnosis and/or treatment of TTR associated disorders, and in methods of detecting TTR. Such anti-TTR antibodies, may include antibodies as in Table 3 below.

Table 3

Anti-TTR antibodies.

[186] Other antibodies against TTR are described in US20190345237, US20160355576, US20160340419, US20160347832, W02020003172, US20190195896, US10344080, WO2015115332, WO2014124334 and US9790269.

B. Non-Human Antibodies

[187] The production of other non-human antibodies, e.g., murine, guinea pig, primate, rabbit or rat, against monomeric TTR or a fragment thereof (e.g, amino acid residues 89-97 or amino acid residues 101-109) can be accomplished by, for example, immunizing the animal with TTR or a fragment thereof. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such an immunogen can be obtained from a natural source, by peptide synthesis, or by recombinant expression. Optionally, the immunogen can be administered fused or otherwise complexed with a carrier protein. Optionally, the immunogen can be administered with an adjuvant. Several types of adjuvant can be used as described below. Complete Freund’s adjuvant followed by incomplete adjuvant is preferred for immunization of laboratory animals. Rabbits or guinea pigs are typically used for making polyclonal antibodies. Mice are typically used for making monoclonal antibodies. Antibodies are screened for specific binding to monomeric TTR or an epitope within TTR (e.g, an epitope comprising one or more of amino acid residues 89-97 or of amino acid residues 101-109). Such screening can be accomplished by determining binding of an antibody to a collection of monomeric TTR variants, such as TTR variants containing amino acid residues 89-97 amino acid residues 101-109 or mutations within these residues, and determining which TTR variants bind to the antibody. Binding can be assessed, for example, by Western blot, FACS or ELISA.

C. Humanized Antibodies

[188] A humanized antibody is a genetically engineered antibody in which CDRs from a non human “donor” antibody are grafted into human “acceptor” antibody sequences (see, e.g,

Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557). The acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having at least three, four, five or all CDRs entirely or substantially from a donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences. Similarly a humanized heavy chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody heavy chain, and a heavy chain variable region framework sequence and heavy chain constant region, if present, substantially from human heavy chain variable region framework and constant region sequences. Similarly a humanized light chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody light chain, and a light chain variable region framework sequence and light chain constant region, if present, substantially from human light chain variable region framework and constant region sequences. Other than nanobodies and dAbs, a humanized antibody comprises a humanized heavy chain and a humanized light chain.

A CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least 85%, 90%, 95% or 100% of corresponding residues (as defined by any conventional definition but preferably defined by Rabat) are identical between the respective CDRs. The variable region framework sequences of an antibody chain or the constant region of an antibody chain are substantially from a human variable region framework sequence or human constant region respectively when at least 85%, 90%, 95% or 100% of corresponding residues defined by any conventional definition but preferably defined by Rabat are identical. To be classified as humanized under the 2014 World Health Organization (WHO) International non proprietary names (INN) definition of humanized antibodies, an antibody must have at least 85% identity in the mature variable regions to human germline antibody sequences (i.e., prior to somatic hypermutation). Mixed antibodies are antibodies for which one antibody chain (e.g., heavy chain) meets the threshold but the other chain (e.g., light chain) does not meet the threshold. An antibody is classified as chimeric if neither chain meets the threshold, even though the variable framework regions for both chains were substantially human with some murine backmutations. See, Jones et al. (2016) The INNs and outs of antibody nonproprietary names, mAbs 8:1, 1-9, DOI: 10.1080/19420862.2015.1114320. See also “WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review)” (Internet) 2014. Available from: http://www. who.int/medicines/services/inn/BioRev2014.pdf), incorporated herein by reference. For the avoidance of doubt, the term “humanized” as used herein is not intended to be limited to the 2014 WHO INN definition of humanized antibodies. Some of the humanized antibodies provided herein have at least 85% sequence identity to human germline sequences in either or both mature variable regions and some of the humanized antibodies provided herein have less than 85% sequence identity to human germline sequences in either or both mature variable regions. Some of the mature heavy chain variable regions of the humanized antibodies provided herein have from about 60% to 100% sequence identity to human germ line sequences, such as, for example, in the range of about 60% to 69%, 70% to 79%, 80% to 84%, or 85% to 89%. Some of the mature heavy chain variable regions fall below the 2014 WHO INN definition and have, for example, about 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, or 82%, 83%, or 84% sequence identity to human germ line sequences, while other mature heavy chain variable regions meet the 2014 WHO INN definition and have about 85%, 86%, 87%, 88%, 89% or greater sequence identity to human germ line sequences. Some of the mature light chain variable regions of the humanized antibodies provided herein have from about 60% to 100% sequence identity to human germ line sequences, such as, for example, in the range of about 80% to 84% or 85% to 89%. Some of the mature light chain variable regions fall below the 2014 WHO INN definition and have, for example, about 81%, 82%, 83% or 84% sequence identity to human germ line sequences, while other mature light chain variable regions meet the 2014 WHO INN definition and have about 85%, 86%, 87%, 88%, 89% or greater sequence identity to human germ line sequences. Some humanized antibodies provided herein that are "chimeric" under the 2014 WHO INN definition have mature heavy chain variable regions with less than 85% identity to human germ line sequences paired with mature light chain variable regions having less than 85% identity to human germ line sequences. Some humanized antibodies provided herein are "mixed" under the 2014 WHO INN definition, for example, having a mature heavy chain variable region with at least 85% sequence identity to human germ line sequences paired with a mature light chain variable region having less than 85% sequence identity to human germ line sequences, or vice versa. Some humanized antibodies provided herein meet the 2014 WHO INN definition of "humanized" and have a mature heavy chain variable region with at least 85% sequence identity to human germ line sequences paired with a mature light chain variable region having at least 85% sequence identity to human germ line sequences. Exemplary 18C5 antibodies that meet the 2014 WHO INN definition of "humanized" include antibodies having a mature heavy chain variable region with an amino acid sequence of SEQ ID NO:85 or SEQ ID NO: 86 paired with a mature light chain variable region having an amino acid sequence of SEQ ID NO:91 or SEQ ID NO:92.

[189] Although humanized antibodies often incorporate all six CDRs (preferably as defined by Kabat) from a mouse antibody, they can also be made with less than all CDRs ( e.g ., at least 3, 4, or 5 CDRs) from a mouse antibody (e.g., Pascalis etal, J. Immunol. 169:3076, 2002; Vajdos et al, J. of Mol. Biol., 320: 415-428, 2002; Iwahashi et al, Mol. Immunol. 36:1079-1091, 1999; Tamura e/a/, J. Immunol., 164:1432-1441, 2000).

[190] In some antibodies only part of the CDRs, namely the subset of CDR residues required for binding, termed the SDRs, are needed to retain binding in a humanized antibody. CDR residues not contacting antigen and not in the SDRs can be identified based on previous studies (for example residues H60-H65 in CDR H2 are often not required), from regions of Kabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling and/or empirically, or as described in Gonzales et al, Mol. Immunol. 41 : 863, 2004. In such humanized antibodies at positions in which one or more donor CDR residues is absent or in which an entire donor CDR is omitted, the amino acid occupying the position can be an amino acid occupying the corresponding position (by Kabat numbering) in the acceptor antibody sequence. The number of such substitutions of acceptor for donor amino acids in the CDRs to include reflects a balance of competing considerations. Such substitutions are potentially advantageous in decreasing the number of mouse amino acids in a humanized antibody and consequently decreasing potential immunogenicity. However, substitutions can also cause changes of affinity, and significant reductions in affinity are preferably avoided. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically.

[191] The human acceptor antibody sequences can optionally be selected from among the many known human antibody sequences to provide a high degree of sequence identity (e.g, 65-85% identity) between a human acceptor sequence variable region frameworks and corresponding variable region frameworks of a donor antibody chain. [192] An example of an acceptor sequence for the 18C5 heavy chain is the humanized Crenezumab Fab (CreneFab) VH, with PDB accession code 5VZY (SEQ ID NO:83). An example of an acceptor sequence for the 18C5 light chain is the humanized Crenezumab Fab (CreneFab) VL, with PDB accession code 5VZY (SEQ ID NO:89). Another example of an acceptor sequence for the 18C5 light chain is the human germline gene IGKV2-30*02 (SEQ ID NO: 90).

[193] If more than one human acceptor antibody sequence is selected for a chain (either light or heavy), a composite or hybrid of those acceptors can be used for that chain, and the amino acids used at different can be taken from any of the human acceptor antibody sequences used.

[194] Certain amino acids from the human variable region framework residues can be selected for substitution based on their possible influence on CDR conformation and/or binding to antigen. Investigation of such possible influences is by modeling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids.

[195] For example, when an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid can be substituted by the equivalent framework amino acid from the mouse antibody when it is reasonably expected that the amino acid:

(1) noncovalently binds antigen directly;

(2) is adjacent to a CDR region or within a CDR as defined by Chothia but not Rabat;

(3) otherwise interacts with a CDR region ( e.g ., is within about 6 A of a CDR region), (e.g., identified by modeling the light or heavy chain on the solved structure of a homologous known immunoglobulin chain); or

(4) is a residue participating in the VL-VH interface.

[196] The invention provides humanized forms of the murine 18C5 antibody including 2 exemplified humanized heavy chain mature variable regions (hul8C5-VH_vl (SEQ ID NO:85), and hul8C5-VH_v2 (SEQ ID NO:86)), and 2 exemplified humanized light chain mature variable regions (hul8C5-VL_vl (SEQ ID NO:91) and hul8C5-VL_v2 (SEQ ID NO: 92)). [197] In an embodiment, humanized sequences are generated using a two-stage PCR protocol that allows introduction of multiple mutations, deletions, and insertions using QuikChange site- directed mutagenesis [Wang, W. and Malcolm, B.A. (1999) BioTechniques 26:680-682)].

[198] Framework residues from classes (1) through (3) as defined by Queen, US 5,530,101, are sometimes alternately referred to as canonical and vernier residues. Framework residues that help define the conformation of a CDR loop are sometimes referred to as canonical residues (Chothia & Lesk, ./. Mol. Biol. 196:901-917 (1987); Thornton & Martin, J. Mol. Biol. 263:800- 815 (1996)). Framework residues that support antigen-binding loop conformations and play a role in fine-tuning the fit of an antibody to antigen are sometimes referred to as vernier residues (Foote & Winter, J. Mol. £/ø/ 224:487-499 (1992)).

[199] Other framework residues that are candidates for substitution are residues creating a potential glycosylation site. Still other candidates for substitution are acceptor human framework amino acids that are unusual for a human immunoglobulin at that position. These amino acids can be substituted with amino acids from the equivalent position of the mouse donor antibody or from the equivalent positions of more typical human immunoglobulins.

[200] Other framework residues that are candidates for substitution are N-terminal glutamic acid residues (E) that may be replaced with glutamine (Q).

[201] Exemplary humanized antibodies are humanized forms of the mouse 18C5, designated Hul8C5.

[202] The mouse antibody 18C5 comprises mature heavy and light chain variable regions having amino acid sequences comprising SEQ ID NO: 81 and SEQ ID NO:87, respectively. The invention provides 2 exemplified humanized mature heavy chain variable regions: hul8C5-

VH vl and hul8C5-VH_v2. The invention further provides 2 exemplified human mature light chain variable regions: hul8C5-VL_vl and hul8C5-VL_v2.

[203] For reasons such as possible influence on CDR conformation and/or binding to antigen, mediating interaction between heavy and light chains, interaction with the constant region, being a site for desired or undesired post-translational modification, being an unusual residue for its position in a human variable region sequence and therefore potentially immunogenic, getting aggregation potential, and other reasons, the following 8 variable region framework positions of 18C5 were considered as candidates for substitutions in the 2 exemplified human mature light chain variable regions and the 2 exemplified human mature heavy chain variable regions, as further specified in Example 7: L2 (I2V), L45 (Q45R), H37 (V37A), H45 (L45Q), H47 (L47W), H48 (V48I), H49 (A49G), and H94 (S94R).

[204] Here, as elsewhere, the first-mentioned residue is the residue of a humanized antibody formed by grafting Rabat CDRs or a composite Chothia Rabat CDR in the case of CDR-H1 into a human acceptor framework, and the second-mentioned residue is a residue being considered for replacing such residue. Thus, within variable region frameworks, the first mentioned residue is human, and within CDRs, the first mentioned residue is mouse.

[205] Exemplified antibodies include any permutations or combinations of the exemplified mature heavy and light chain variable regions of 18C5 e.g., hul8C5VH_vl/ hul8C5VL_vl, hul8C5VH_vl/ hul8C5VL_v2, hul8C5VH_v2/ hul8C5VL_vl, or hul8C5VH_v2/ hul8C5VL_v2.

[206] The invention provides variants of the 18C5 humanized antibody in which the humanized mature heavy chain variable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of hul8C5-VH_vl, and hul8C5-VH_v2. (SEQ ID NOs: 85-86) and the humanized mature light chain variable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of hul8C5-VL_vl, and hul8C5-VL_v2, (SEQ ID NOs: 91-92). In some such antibodies at least 1, 2, 3, 4, 5, 6, 7, or all 8, of the backmutations or other mutations found in SEQ ID NO: 86 and SEQ ID NO: 92 are retained.

[207] In some humanized 18C5 antibodies, at least one of the following positions is occupied by the amino acid as specified: H37 is occupied by V or A, H45 is occupied by L or Q, H47 is occupied by L or W, H48 is occupied by L or I, H49 is occupied by A or G, and H94 is occupied by S or R.

[208] In some humanized 18C5 antibodies, positions H37, H45, H47, H48, H49, and H94 in the VH region are occupied by A, Q, W, I, G, and R, respectively, as in hul8C5-VH_v2. [209] In some humanized 18C5 antibodies, at least one of the following positions is occupied by the amino acid as specified: L2 is occupied by I or V and L45 is occupied by Q or R.

[210] In some humanized 18C5 antibodies, positions L2 and L45 in the VL region are occupied by V and R, respectively, as in hul8C5-VL_v2.

[211] In some humanized 18C5 antibodies, the variable heavy chain has > 85% identity to human sequence. In some humanized 18C5 antibodies, the variable light chain has > 85% identity to human sequence. In some humanized 18C5 antibodies, each of the variable heavy chain and variable light chain has > 85% identity to human germline sequence.

[212] The CDR regions of such humanized antibodies can be identical or substantially identical to the CDR regions of the 9D5 or 18C5 mouse donor antibody. The CDR regions can be defined by any conventional definition, such as those in Table 1, but are preferably as defined by Rabat or Kabat+Chothia composite.

[213] Variable regions framework positions are in accordance with Rabat numbering unless otherwise stated. Other such variants typically differ from the sequences of the exemplified Hul8C5 heavy and light chains by a small number ( e.g typically no more than 1, 2, 3, 5, 10, or 15) of replacements, deletions or insertions.

[214] A possibility for additional variation in humanized 9D5 or 18C5 variants is additional backmutations in the variable region frameworks. Many of the framework residues not in contact with the CDRs in the humanized mAh can accommodate substitutions of amino acids from the corresponding positions of the donor mouse mAh or other mouse or human antibodies, and even many potential CDR-contact residues are also amenable to substitution. Even amino acids within the CDRs may be altered, for example, with residues found at the corresponding position of the human acceptor sequence used to supply variable region frameworks. In addition, alternate human acceptor sequences can be used, for example, for the heavy and/or light chain.

If different acceptor sequences are used, one or more of the backmutations recommended above may not be performed because the corresponding donor and acceptor residues are already the same without backmutations. [215] Some replacements or backmutations in Hu9D5 or Hul8C5variants (whether or not conservative) have no substantial effect on the binding affinity or potency of the humanized mAb, that is, its ability to bind to monomeric TTR ( e.g ., the potency in some or all of the assays described in the present examples of the variant humanized 9D5 or 18C5 antibody is essentially the same, i.e., within experimental error, as that of murine 9D5 or murine 18C5). Exemplary humanized 9D5 variants are described in WO 2016/120810.

D. Chimeric and Veneered Antibodies

[216] Some methods of the invention use chimeric and veneered forms of non-human antibodies, particularly the 9D5 or 18C5 antibodies of the examples.

[217] A chimeric antibody is an antibody in which the mature variable regions of light and heavy chains of a non-human antibody (e.g., a mouse) are combined with human light and heavy chain constant regions. Such antibodies substantially or entirely retain the binding specificity of the mouse antibody, and are about two-thirds human sequence. In an embodiment, a chimeric 18C5 antibody has a mature heavy chain variable region amino acid sequence of SEQ ID NO:81, a mature light chain variable region amino acid sequence of SEQ ID NO:87, a human heavy chain constant region amino acid sequence of SEQ ID NO: 17, and a human light chain constant region amino acid sequence of SEQ ID NO: 19. An exemplary chimeric 9D5 antibody is described in WO 2016/120810,

[218] A veneered antibody is a type of humanized antibody that retains some and usually all of the CDRs and some of the non-human variable region framework residues of a non-human antibody but replaces other variable region framework residues that may contribute to B- or T- cell epitopes, for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) with residues from the corresponding positions of a human antibody sequence. The result is an antibody in which the CDRs are entirely or substantially from a non-human antibody and the variable region frameworks of the non-human antibody are made more human-like by the substitutions. Veneered forms of the 9D5 or 18C5 antibody are included in the invention.

E. Human Antibodies [219] Human antibodies against monomeric TTR or a fragment thereof ( e.g ., amino acid residues 89-97 (SEQ ID NO:45) of TTR, or amino acid residues 101-109 (SEQ ID NO:30) of TTR) are provided by a variety of techniques described below. Some human antibodies are selected by competitive binding experiments, by the phage display method of Winter, above, or otherwise, to have the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the examples. Human antibodies can also be screened for particular epitope specificity by using only a fragment of TTR, such as a TTR variant containing only amino acid residues 89-97 or amino acid residues 101-109 of TTR, as the target antigen, and/or by screening antibodies against a collection of TTR variants, such as TTR variants containing various mutations within amino acid residues 89-97 or amino acid residues 101-109 of TTR.

[220] Methods for producing human antibodies include the trioma method of Oestberg et al, Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634,664; and Engleman et al., US Patent 4,634,666, use of transgenic mice including human immunoglobulin genes (see, e.g., Lonberg et al, W093/12227 (1993); US 5,877,397; US 5,874,299; US 5,814,318; US 5,789,650; US 5,770,429; US 5,661,016; US 5,633,425; US 5,625,126; US 5,569,825; US 5,545,806; Neuberger, Nat. Biotechnol. 14:826 (1996); and Kucherlapati, WO 91/10741 (1991)) and phage display methods (see, e.g., Dower et al, WO 91/17271; McCafferty etal, WO 92/01047; US 5,877,218; US 5,871,907; US 5,858,657; US 5,837,242; US 5,733,743; and US 5,565,332).

F. Selection of Constant Region

[221] The heavy and light chain variable regions of chimeric, veneered or humanized antibodies can be linked to at least a portion of a human constant region. The choice of constant region depends, in part, whether antibody-dependent cell-mediated cytotoxicity, antibody dependent cellular phagocytosis and/or complement dependent cytotoxicity are desired. For example, human isotypes IgGl and IgG3 have complement-dependent cytotoxicity and human isotypes IgG2 and IgG4 do not. Human IgGl and IgG3 also induce stronger cell mediated effector functions than human IgG2 and IgG4. Light chain constant regions can be lambda or kappa. Numbering conventions for constant regions include EU numbering (Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)), Rabat numbering (Rabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1991, IMGT unique numbering (Lefranc M.-P. et al., IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains, Dev. Comp. Immunol., 29, 185- 203 (2005), and IMGT exon numbering (Lefranc, supra).

[222] One or several amino acids at the amino or carboxy terminus of the light and/or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al, US Patent No. 5,624,821; Tso et al., US Patent No. 5,834,597; and Lazar et al, Proc. Natl Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g, Hinton et al., J.

Biol. Chem. 279:6213, 2004). Exemplary substitutions include a Gin at position 250 and/or a Leu at position 428 (EU numbering is used in this paragraph for the constant region) for increasing the half-life of an antibody. Substitution at any or all of positions 234, 235, 236 and/or 237 reduce affinity for Fey receptors, particularly FcyRI receptor (see, e.g, US

6.624.821). An alanine substitution at positions 234, 235, and 237 of human IgGl can be used for reducing effector functions. Some antibodies have alanine substitution at positions 234, 235 and 237 of human IgGl for reducing effector functions. Optionally, positions 234, 236 and/or 237 in human IgG2 are substituted with alanine and position 235 with glutamine (see, e.g, US

5.624.821). In some antibodies, a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 by EU numbering of human IgGl is used. In some antibodies, a mutation at one or more of positions 318, 320, and 322 by EU numbering of human IgGl is used. In some antibodies, positions 234 and/or 235 are substituted with alanine and/or position 329 is substituted with glycine. In some antibodies, positions 234 and 235 are substituted with alanine, such as in SEQ ID NO:23. In some antibodies, the isotype is human IgG2 or IgG4.

[223] An exemplary human light chain kappa constant region has the amino acid sequence of SEQ ID NO:24. The N-terminal arginine of SEQ ID NO:24 can be omitted, in which case light chain kappa constant region has the amino acid sequence of SEQ ID NO:25. An exemplary human IgGl heavy chain constant region has the amino acid sequence of SEQ ID NO:21 (with or without the C-terminal lysine). Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab', F(ab')2, and Fv, or as single chain antibodies in which heavy and light chain mature variable domains are linked through a spacer.

[224] Human constant regions show allotypic variation and isoallotypic variation between different individuals, that is, the constant regions can differ in different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera recognizing an isoallotype bind to a non-polymorphic region of a one or more other isotypes. Thus, for example, another heavy chain constant region is of IgGl Glm3 allotype and has the amino acid sequence of SEQ ID NO:22. Another heavy chain constant region of the IgGl Glm3 allotype has the amino acid sequence of SEQ ID NO:23 (with or without the C-terminal lysine). Reference to a human constant region includes a constant region with any natural allotype or any permutation of residues occupying positions in natural allotypes.

(7. Expression of Recombinant Antibodies

[225] A number of methods are known for producing chimeric and humanized antibodies using an antibody-expressing cell line ( e.g hybridoma). For example, the immunoglobulin variable regions of antibodies can be cloned and sequenced using well known methods. In one method, the heavy chain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells. Consensus primers are employed to the VH region leader peptide encompassing the translation initiation codon as the 5' primer and a g2b constant regions specific 3' primer. Exemplary primers are described in U.S. patent publication US 2005/0009150 by Schenk et al. (hereinafter “Schenk”). The sequences from multiple, independently derived clones can be compared to ensure no changes are introduced during amplification. The sequence of the VH region can also be determined or confirmed by sequencing a VH fragment obtained by 5' RACE RT-PCR methodology and the 3' g2b specific primer.

[226] The light chain variable VL region can be cloned in an analogous manner. In one approach, a consensus primer set is designed for amplification of VL regions using a 5’ primer designed to hybridize to the VL region encompassing the translation initiation codon and a 3' primer specific for the Ck region downstream of the V-J joining region. In a second approach, 5'RACE RT-PCR methodology is employed to clone a VL encoding cDNA. Exemplary primers are described in Schenk, supra. The cloned sequences are then combined with sequences encoding human (or other non-human species) constant regions. Exemplary sequences encoding human constant regions include SEQ ID NO:32, which encodes a human IgGl constant region, and SEQ ID NOs:33 and 34, which encode a human kappa light chain constant region.

[227] In one approach, the heavy and light chain variable regions are re-engineered to encode splice donor sequences downstream of the respective VDJ or VJ junctions and are cloned into a mammalian expression vector, such as pCMV- hyl for the heavy chain and pCMV-Mcl for the light chain. These vectors encode human gΐ and Ck constant regions as exonic fragments downstream of the inserted variable region cassette. Following sequence verification, the heavy chain and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies. Conditioned media is collected 48 hours post-transfection and assayed by western blot analysis for antibody production or ELISA for antigen binding. The chimeric antibodies are humanized as described above.

[228] Chimeric, veneered, humanized, and human antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequences of antibody chains, including naturally associated or heterologous expression control elements, such as a promoter. The expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and the collection and purification of the crossreacting antibodies.

[229] These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., ampicillin resistance or hygromycin resistance, to permit detection of those cells transformed with the desired DNA sequences.

[230] E. coli is one prokaryotic host useful for expressing antibodies, particularly antibody fragments. Microbes, such as yeast, are also useful for expression. Saccharomyces is a yeast host with suitable vectors having expression control sequences, an origin of replication, termination sequences, and the like as desired. Typical promoters include 3 -phosphogly cerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

[231] Mammalian cells can be used for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed, and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2/0 and NSO. The cells can be nonhuman. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al, Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al, J. Immunol. 148:1149 (1992).

[232] Alternatively, antibody coding sequences can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., U.S. Pat. No. 5,741,957; Ti.S. Pat. No. 5,304,489; and U.S. Pat. No. 5,849,992). Suitable transgenes include coding sequences for light and/or heavy chains operably linked with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin.

[233] The vectors containing the DNA segments of interest can be transferred into the host cell by methods depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics, or viral-based transfection can be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. For production of transgenic animals, transgenes can be microinjected into fertilized oocytes or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes. [234] Having introduced vector(s) encoding antibody heavy and light chains into cell culture, cell pools can be screened for growth productivity and product quality in serum -free media. Top-producing cell pools can then be subjected of FACS-based single-cell cloning to generate monoclonal lines. Specific productivities above 50 pg or 100 pg per cell per day, which correspond to product titers of greater than 7.5 g/L culture, can be used. Antibodies produced by single cell clones can also be tested for turbidity, filtration properties, PAGE, IEF, UV scan, HP- SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assay, such as ELISA or Biacore. A selected clone can then be banked in multiple vials and stored frozen for subsequent use.

[235] Once expressed, antibodies can be purified according to standard procedures of the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[236] Methodology for commercial production of antibodies can be employed, including codon optimization, selection of promoters, selection of transcription elements, selection of terminators, serum-free single cell cloning, cell banking, use of selection markers for amplification of copy number, CHO terminator, or improvement of protein titers (see, e.g, US 5,786,464; US 6,114,148; US 6,063,598; US 7,569,339; W02004/050884; W02008/012142; W02008/012142; W02005/019442; W02008/107388; W02009/027471; and US 5,888,809).

H. Antibody Screening Assays

[237] Antibodies can be subject to several screens including binding assays, functional screens, screens in animal models of diseases associated with TTR deposits, and clinical trials. Binding assays test for specific binding and, optionally, affinity and epitope specificity to monomeric TTR or a fragment thereof. For example, binding assays can screen for antibodies that bind to amino acid residues 89-97 (SEQ ID NO:45) or amino acid residues 101-109 (SEQ ID NO:30) of TTR , which are epitopes that are buried in the native TTR tetramer and exposed in monomeric, misfolded, aggregated, or fibril forms of TTR. Antibodies can also be screened for the ability to bind pre-fibrillar, non-native conformations of TTR and TTR amyloid fibrils but not native TTR conformations. For example, antibodies can be screened for the ability to bind to monomeric forms of TTR created by dissociation or disaggregation of native tetrameric TTR, and can be counter-screened against native tetrameric TTR, as described in the examples or otherwise. Likewise, antibodies can also be screened for their immunoreactivity on TTR-mediated amyloidosis tissue but not on healthy tissue. Such screens are sometimes performed in competition with an exemplary antibody, such as an antibody having the variable regions of 9D5 or of 18C5 or IgGl kappa isotype. Optionally, either the antibody or TTR target is immobilized in such assay.

[238] Functional assays can be performed in cellular models including cells naturally expressing TTR or transfected with DNA encoding TTR or a fragment thereof. Suitable cells include cells derived from cardiac tissue or other tissues affected by TTR amyloidogenesis.

Cells can be screened for reduced levels of monomeric, misfolded, aggregated, or fibril forms of TTR ( e.g by Western blotting or immunoprecipitation of cell extracts or supernatants) or reduced toxicity attributable to monomeric, misfolded, aggregated, or fibril forms of TTR. For example, antibodies can be tested for the ability to inhibit or reduce aggregation of TTR, inhibit or reduce TTR fibril formation, reduce TTR deposits, clear aggregated TTR, or stabilize non toxic conformations of TTR.

[239] Other functional assays can be performed in solution, such as testing whether an antibody is capable of disrupting or reducing TTR fibril formation when monomeric TTR or misfolded TTR intermediates in solution are contacted with the antibody. The extent of fibril formation can be probed by turbidity measurements, for example, at 400 nm on a UV-visible spectrometer equipped with a temperature control unit. Thioflavin-T can also be used to assess the extent of amyloid fibril formation. For example, a five-fold molar excess of Thioflavin-T can be added to TTR samples and left at room temperature for 30 minutes before measurements are taken. Thioflavin-T fluorescence can be monitored using a spectrofluorimeter. See US 2014/0056904.

[240] Animal model screens test the ability of the antibody to therapeutically or prophylactically treat signs and/or symptoms in an animal model simulating a human disease associated with accumulation of TTR or TTR deposits. Such diseases include types of TTR amyloidosis, such as wild-type ATTR amyloidosis (also called senile systemic amyloidosis SSA), senile cardiac amyloidosis (SCA), familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), and central nervous system selective amyloidosis (CNSA). Suitable signs and/or symptoms that can be monitored include the presence and extent of amyloid deposits in various tissues, such as the gastrointestinal tract or heart. The extent of reduction of amyloid deposits can be determined by comparison with an appropriate control, such the level of TTR amyloid deposits in control animals that have received a control antibody (e.g, an isotype matched control antibody), a placebo, or no treatment at all. An exemplary animal model for testing activity against a TTR amyloidosis is a mouse model carrying a null mutation at the endogenous mouse Ttr locus and the human mutant TTR gene comprising a V30M mutation that is associated with familial amyloidotic polyneuropathy. See, e.g, Kohno et al, Am. J. Path. 150(4): 1497-1508 (1997); Cardoso and Saraiva, FASEB J 20(2):234-239 (2006). Similar models also exist, including other models for familial versions of TTR amyloidosis and models for sporadic versions of TTR amyloidosis. See, e.g, Teng et al, Lab. Invest. 81(3): 385-396 (2001); Ito and Maeda, Mouse Models of Transthyretin Amyloidosis, in Recent Advances in Transthyretin Evolution, Structure, and Biological Functions, pp. 261-280 (2009) (Springer Berlin Heidelberg). Transgenic animals can include a human TTR transgene, such as a TTR transgene with a mutation associated with TTR amyloidosis or a wild-type TTR transgene. To facilitate testing in animal models, chimeric antibodies having a constant region appropriate for the animal model can be used (e.g, mouse-rat chimeras could be used for testing antibodies in rats). It can be concluded that a humanized version of an antibody will be effective if the corresponding mouse antibody or chimeric antibody is effective in an appropriate animal model and the humanized antibody has similar binding affinity (e.g, within experimental error, such as by a factor of 1.5, 2, or 3).

[241] Clinical trials test for safety and efficacy in a human having a disease associated with TTR amyloidosis.

I. Conjugated Antibodies

[242] Conjugated antibodies that specifically bind to antigens exposed in pathogenic forms of TTR but not in native tetrameric form of TTR, such as amino acid residues 89-97 (SEQ ID NO:45) or amino acid residues 101-109 (SEQ ID NO:30) of TTR, are useful in detecting the presence of monomeric, misfolded, aggregated, or fibril forms of TTR; monitoring and evaluating the efficacy of therapeutic agents being used to treat subjects diagnosed with a TTR amyloidosis; inhibiting or reducing aggregation of TTR; inhibiting or reducing TTR fibril formation; reducing or clearing TTR deposits; stabilizing non-toxic conformations of TTR; or treating or effecting prophylaxis of a TTR amyloidosis in a subject. For example, such antibodies can be conjugated with other therapeutic moieties, other proteins, other antibodies, and/or detectable labels. See WO 03/057838; US 8,455,622.

[243] Conjugated therapeutic moieties can be any agent that can be used to treat, combat, ameliorate, prevent, or improve an unwanted condition or disease in a subject, such as a TTR amyloidosis. Therapeutic moieties can include, for example, immunomodulators or any biologically active agents that facilitate or enhance the activity of the antibody. An immunomodulator can be any agent that stimulates or inhibits the development or maintenance of an immunologic response. If such therapeutic moieties are coupled to an antibody specific for monomeric, misfolded, aggregated, or fibril forms of TTR, such as the antibodies described herein, the coupled therapeutic moieties will have a specific affinity for non-native, pathogenic forms of TTR over native tetrameric form of TTR. Consequently, administration of the conjugated antibodies directly targets tissues comprising pathogenic forms of TTR with minimal damage to surrounding normal, healthy tissue. This can be particularly useful for therapeutic moieties that are too toxic to be administered on their own. In addition, smaller quantities of the therapeutic moieties can be used.

[244] Examples of suitable therapeutic moieties include drugs that reduce levels of TTR, stabilize the native tetrameric structure of TTR, inhibit aggregation of TTR, disrupt TTR fibril or amyloid formation, or counteract cellular toxicity. See, e.g., Almeida and Saraiva, FEBS Letters 586:2891-2896 (2012); Saraiva, FEBS Letters 498:201-203 (2001); Ando et ah, Orphanet Journal of Rare Diseases 8:31 (2013); Ruberg and Berk, Circulation 126:1286-1300 (2012); Johnson etal., J. Mol. Biol. 421(2-3): 185-203 (2012, Ueda and Ando, Translational Neurodegeneration 3:19 (2014), and Hawkins et al. Annals of Medicine 47:625-638 (2015)). For example, antibodies can be conjugated to tafamidis, diflunisal, AGIO, ALN-TTROl,

ALNTTR02, antisense oligonucleotides such as IONIS TTRRx (inotersen), siRNAs such as patisiran or revusiran, doxycycline (doxy), tauroursodeoxycholic acid (TUDCA), Doxy- TUDCA, cyclodextrin (CyD), 4'-iodo-4'-deoxydoxorubicin (IDOX), epigallocatechin gallate (EGCG), curcumin, resveratrol (3,5,4’-trihydroxystilbene), or antibodies to serum amyloid P component (SAP). Other representative therapeutic moieties include other agents known to be useful for treatment, management, or amelioration of a TTR amyloidosis or signs and/or symptoms of a TTR amyloidosis. See, e.g., Ando et al, Orphanet Journal of Rare Diseases 8:31 (2013) for common clinical signs and/or symptoms of TTR amyloidosis and typical agents used to treat those signs and/or symptoms.

[245] Antibodies can also be coupled with other proteins. For example, antibodies can be coupled with Fynomers. Fynomers are small binding proteins (e.g, 7 kDa) derived from the human Fyn SH3 domain. They can be stable and soluble, and they can lack cysteine residues and disulfide bonds. Fynomers can be engineered to bind to target molecules with the same affinity and specificity as antibodies. They are suitable for creating multi-specific fusion proteins based on antibodies. For example, Fynomers can be fused to N-terminal and/or C- terminal ends of antibodies to create bi- and tri-specific FynomAbs with different architectures. Fynomers can be selected using Fynomer libraries through screening technologies using FACS, Biacore, and cell-based assays that allow efficient selection of Fynomers with optimal properties. Examples of Fynomers are disclosed in Grabulovski et al, J. Biol. Chem. 282:3196-3204 (2007); Bertschinger etal, Protein Eng. Des. Sel. 20:57-68 (2007); Schlatter et al, MAbs. 4:497-508 (2011); Banner etal, Acta. Crystallogr. D. Biol. Crystallogr. 69(Pt6): 1124-1137 (2013); and Brack et al, Mol. Cancer Ther. 13:2030-2039 (2014).

[246] The antibodies disclosed herein can also be coupled or conjugated to one or more other antibodies (e.g, to form antibody heteroconjugates). Such other antibodies can bind to different epitopes within TTR or a portion thereof or can bind to a different target antigen. Such anti-TTR antibodies binding to TTR epitopes different from that of 9D5 or 18C5, may include antibodies as in Table 3.

[247] Antibodies can also be coupled with a detectable label. Such antibodies can be used, for example, for diagnosing a TTR amyloidosis, for monitoring progression of a TTR amyloidosis, and/or for assessing efficacy of treatment. Such antibodies are particularly useful for performing such determinations in subjects having or being susceptible to a TTR amyloidosis, or in appropriate biological samples obtained from such subjects. Representative detectable labels that may be coupled or linked to an antibody disclosed herein include various enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such streptavidin, avidin or biotin; fluorescent materials, such as umbelliferone, DyLight fluors, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as luminol; bioluminescent materials, such as luciferase, luciferin, and aequorin; radioactive materials, such as yttrium⁹⁰ (90Y), radiosilver-111, radiosilver- 199, Bismuth²¹³, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I,), carbon (¹⁴C), sulfur (⁵S), tritium (¾), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ^U1ln,), technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷RU, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵ Sr, ³²P, ¹⁵³Gd, ¹⁶⁹ Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵ Se, ¹¹³Sn, and ¹¹⁷Tin; positron emitting metals using various positron emission tomographies; nonradioactive paramagnetic metal ions; and molecules that are radiolabelled or conjugated to specific radioisotopes. Representative detectable labels that may be coupled or linked to an antibody disclosed herein include electrochemiluminescent labels, for example MSD GOLD SULFO- TAG NHS-Ester (SULFO-TAG) (Meso Scale Diagnostics, Rockville, MD).

[248] Linkage of radioisotopes to antibodies may be performed with conventional bifunction chelates. For radiosilver- 111 and radiosilver- 199 linkage, sulfur-based linkers may be used. See Hazra et ah, Cell Biophys. 24-25:1-7 (1994). Linkage of silver radioisotopes may involve reducing the immunoglobulin with ascorbic acid. For radioisotopes such as 11 lln and 90Y, ibritumomab tiuxetan can be used and will react with such isotopes to form 11 lln-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl LS91-S95 (2001).

[249] Therapeutic moieties, other proteins, other antibodies, and/or detectable labels may be coupled or conjugated, directly or indirectly through an intermediate ( e.g ., a linker), to a murine, chimeric, veneered, or humanized 9D5 or 18C5 antibody using techniques known in the art. See e.g., Amon etal, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld etal. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et ah, "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson etal. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera etal. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and Thorpe et ah, Immunol. Rev., 62: 119-58 (1982). Suitable linkers include, for example, cleavable and non-cleavable linkers. Different linkers that release the coupled therapeutic moieties, proteins, antibodies, and/or detectable labels under acidic or reducing conditions, on exposure to specific proteases, or under other defined conditions can be employed.

VI. Therapeutic Applications

[250] The above antibodies can be used for treating or effecting prophylaxis of a disease in a subject having or at risk for the disease mediated at least in part by transthyretin (TTR), and particularly by monomeric, misfolded, aggregated, or fibril forms of TTR in combination with monitoring methods. Although an understanding of mechanism is not required for practice, it is believed that any or all of the following mechanisms may contribute to treatment of TTR amyloidosis using the above antibodies: antibody-mediated inhibition of TTR aggregation and fibril formation, antibody-mediated stabilization of non-toxic conformations of TTR ( e.g ., tetrameric forms), or antibody-mediated clearance of aggregated TTR, oligomeric TTR, or monomeric TTR. Antibody-drug conjugates can have additional mechanisms of action determined by the conjugated moiety.

[251] Antibodies are administered in an effective regimen meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of a disorder being treated. If a subject is already suffering from a disorder, the regimen can be referred to as a therapeutically effective regimen. If the subject is at elevated risk of the disorder relative to the general population but is not yet experiencing signs and/or symptoms, the regimen can be referred to as a prophylactically effective regimen. In some instances, therapeutic or prophylactic efficacy can be observed in an individual subject relative to historical controls or past experience in the same subject. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated subjects relative to a control population of untreated subjects. [252] The frequency of administration depends on the half-life of the antibody in the circulation, the condition of the subject and the route of administration among other factors. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the subject’s condition or progression of the disorder being treated. An exemplary frequency for intravenous administration is between weekly and quarterly over a continuous cause of treatment, although more or less frequent dosing is also possible. For subcutaneous administration, an exemplary dosing frequency is daily to monthly, although more or less frequent dosing is also possible.

[253] The number of dosages administered depends on whether the disorder is acute or chronic and the response of the disorder to the treatment. For acute disorders or acute exacerbations of a chronic disorder, between 1 and 10 doses are often sufficient. Sometimes a single bolus dose, optionally in divided form, is sufficient for an acute disorder or acute exacerbation of a chronic disorder. Treatment can be repeated for recurrence of an acute disorder or acute exacerbation. For chronic disorders, an antibody can be administered at regular intervals, e.g., weekly, fortnightly, monthly, quarterly, every six months for at least 1, 5 or 10 years, or the life of the subject.

VII. Pharmaceutical Compositions and Methods of Use

[254] Provided herein are several methods of diagnosing, monitoring, treating or effecting prophylaxis of diseases or conditions mediated at least in part by transthyretin (TTR), and particularly by monomeric, misfolded, aggregated, or fibril forms of TTR (e.g, TTR amyloidosis). Examples of such diseases include familial TTR amyloidoses, such as familial amyloid cardiomyopathy (FAC), familial amyloid polyneuropathy (FAP), or central nervous system selective amyloidosis (CNSA), and sporadic TTR amyloidoses, such as senile systemic amyloidosis (SSA) or senile cardiac amyloidosis (SCA). Antibodies described above can be incorporated into a pharmaceutical composition for use in such methods. In general, an antibody or pharmaceutical composition containing an antibody is administered to a subject in need thereof. Patients amenable to treatment include individuals at risk of TTR amyloidosis but not showing signs and/or symptoms, as well as subjects presently showing signs and/or symptoms. Some subjects can be treated during the prodromal stage of TTR amyloidosis. [255] The pharmaceutical compositions can be administered prophylactically to individuals who have a known genetic risk of TTR amyloidosis. Such individuals include those having relatives who have experienced such a disease, and those whose risk is determined by analysis of genetic or biochemical markers ( e.g ., mutations in TTR associated with TTR amyloidosis), including using the diagnostic methods provided herein. For example, there are over 100 mutations in the gene encoding TTR that have been implicated in TTR amyloidosis. See, e.g.,

US 2014/0056904; Saraiva, Hum. Mutat. 17(6):493-503 (2001); Damas and Saraiva, J. Struct. Biol. 130:290-299; Dwulet and Benson, Biochem. Biophys. Res. Commun. 114:657-662 (1983).

[256] Individuals suffering from TTR amyloidosis can sometimes be recognized from signs and/or symptoms of TTR amyloidosis as disclosed further below, cardiac disease characterized by thickened ventricular walls in the absence of hypertension; and advanced atrio-ventricular block of unknown origin, particularly when accompanied by a thickened heart; and (6) vitreous body inclusions of the cotton-wool type. See Ando et ah, Or phanet Journal of Rare Diseases 8:31 (2013). Definitive diagnosis of TTR amyloidosis, however, typically relies on target organ biopsies, followed by histological staining of the excised tissue with the amyloid-specific dye, Congo red. If a positive test for amyloid is observed, immunohistochemical staining and mass spectroscopic identification of TTR are subsequently performed to ensure that the precursor protein responsible for amyloid formation is indeed TTR. For familial forms of the diseases, demonstration of a mutation in the gene encoding TTR is then needed before a definitive diagnosis can be made.

[257] The identification of the subject can occur in a clinical setting, or elsewhere, such as in the subject's home, for example, through the subject's own use of a self-testing kit. For example, the subject can be identified based on various signs and/or symptoms such as peripheral neuropathy (sensory and motor), autonomic neuropathy, gastrointestinal impairment, cardiomyopathy, nephropathy, or ocular deposition. See Ando etal, Orphanet Journal of Rare Diseases 8:31 (2013). The subject can also be identified by increased levels of non-native forms of TTR in plasma samples from the subject compared to control samples, as disclosed in the examples. [258] As warranted by family history, genetic testing, or medical screening for TTR amyloidosis, treatment can begin at any age ( e.g ., 20, 30, 40, 50, 60, or 70 years of age). Treatment typically entails multiple dosages over a period of time and can be monitored by assaying antibody or activated T-cell or B-cell responses to a therapeutic agent (e.g., a truncated form of TTR comprising amino acid residues 89-97 or comprising amino acid residues 101-109) over time. If the response falls, a booster dosage is indicated.

[259] In prophylactic applications, an antibody or a pharmaceutical composition of the same is administered to a subject susceptible to, or otherwise at risk of a disease (e.g., TTR amyloidosis) in a regimen (dose, frequency and route of administration) effective to reduce the risk, lessen the severity, or delay the onset of at least one sign or symptom of the disease. In therapeutic applications, an antibody or immunogen to induce an antibody is administered to a subject suspected of, or already suffering from a disease (e.g, TTR amyloidosis) in a regimen (dose, frequency and route of administration) effective to ameliorate or at least inhibit further deterioration of at least one sign or symptom of the disease.

[260] A regimen is considered therapeutically or prophylactically effective if an individual treated subject achieves an outcome more favorable than the mean outcome in a control population of comparable subjects not treated by methods disclosed herein, or if a more favorable outcome is demonstrated for a regimen in treated subjects versus control subjects in a controlled clinical trial (e.g, a phase II, phase II/III, or phase III trial) or an animal model at the p < 0.05 or 0.01 or even 0.001 level.

[261] An effective regimen of an antibody can be used for, e.g, inhibiting or reducing aggregation of TTR in a subject having or at risk of a condition associated with TTR accumulation; inhibiting or reducing TTR fibril formation in a subject having or at risk of a condition associated with TTR accumulation; reducing or clearing TTR deposits or aggregated TTR in a subject having or at risk of a condition associated with TTR accumulation; stabilizing non-toxic conformations of TTR in a subject having or at risk of a condition associated with TTR accumulation; inhibiting toxic effects of TTR aggregates, fibrils or deposits in a subject having or at risk of a condition associated with TTR accumulation; diagnosing the presence or absence of TTR amyloid accumulation in a tissue suspected of comprising the amyloid accumulation; determining a level of TTR deposits in a subject by detecting the presence of bound antibody in the subject following administration of the antibody; detecting the presence of monomeric, misfolded, aggregated, or fibril forms of TTR in a subject; monitoring and evaluating the efficacy of therapeutic agents being used to treat subjects diagnosed with a TTR amyloidosis; inducing an immune response comprising antibodies to TTR in a subject; delaying the onset of a condition associated with TTR amyloid accumulation in a subject; or treating or effecting prophylaxis of a TTR amyloidosis in a subject.

[262] Effective doses vary depending on many different factors, such as means of administration, target site, physiological state of the subject, whether the subject is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.

[263] An exemplary dose range for antibodies can be from about 0.1-20, or 0.5-5 mg/kg body weight ( e.g 0.5, 1, 2, 3, 4 or 5 mg/kg) or 10-1500 mg as a fixed dosage. The dosage depends on the condition of the subject and response to prior treatment, if any, whether the treatment is prophylactic or therapeutic and whether the disorder is acute or chronic, among other factors.

[264] Antibody can be administered in such doses daily, on alternative days, weekly, fortnightly, monthly, quarterly, or according to any other schedule determined by empirical analysis. An exemplary treatment entails administration in multiple doses over a prolonged period, for example, of at least six months. Additional exemplary treatment regimens entail administration once per every two weeks or once a month or once every 3 to 6 months.

[265] Antibodies can be administered via a peripheral route. Routes of administration include topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intranasal or intramuscular. Routes for administration of antibodies can be intravenous or subcutaneous. Intravenous administration can be, for example, by infusion over a period such as 30-90 min. This type of injection is most typically performed in the arm or leg muscles. In some methods, agents are injected directly into a particular tissue where deposits have accumulated, for example intracranial injection.

[266] Pharmaceutical compositions for parenteral administration can be sterile and substantially isotonic (250-350 mOsm/kg water) and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dose form ( i.e ., the dose for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, antibodies can be formulated in aqueous solutions, e.g., in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection). The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[267] The regimens can be administered in combination with, concomitantly with, or sequentially with another agent effective in treatment or prophylaxis of the disease being treated. Such agents can include siRNA to inhibit expression of TTR or Vyndaqel, a stabilizer of TTR in tetramer formation. Such agents can include TTR tetramer stabilizers such as tafamidis or difhmisal (see, .e.g., WO2011116123, US Patent No. 9,150,489), gene therapies to suppress TTR expression such as antisense oligonucleotides such as IONIS-TTRRx (inotersen) (see, e.g., U.S. Patent Nos. 8,101,743, 8,697,860, 9,061,044, and 9,399,774; Japanese Patent No. JP5896175) or siRNAs such as patisiran or revusiran (see, e.g., WO2016033326), amyloid degrader compounds such as doxycycline (doxy), tauroursodeoxycholic acid (TUDCA), Doxy-TUDCA, cyclodextrin (CyD), 4'-iodo-4'-deoxydoxorubicin (IDOX), or antibodies to serum amyloid P component (SAP).

[268] Another agent effective in treatment or prophylaxis of the disease being treated may be administered to a subject who has previously been treated with an antibody disclosed herein.

The subject treated with another agent effective in treatment or prophylaxis of the disease being treated may no longer be receiving treatment with an antibody disclosed herein.

[269] Treatment with antibodies disclosed herein can be combined with other treatments effective against the disorder being treated. Combination treatments can be formulated together or administered separately. Some examples of treatments useful for combination therapies include a second anti -TTR antibody that binds an epitope different from that of 9D5 or 18C5, for example an antibody as disclosed in Table 3. [270] After treatment, the subject's condition can be evaluated to determine the progress or efficacy of such treatment. Such methods preferably test for changes in TTR amyloid levels or levels of non-native forms of TTR. For example, TTR amyloid levels may be evaluated to determine improvement relative to the subject’s TTR amyloid levels under comparable circumstances prior to treatment. The subject’s TTR amyloid levels can also be compared with control populations under comparable circumstances. The control populations can be similarly afflicted, untreated subjects or normal untreated subjects (among other control subjects). Improvement relative to similarly afflicted, untreated subjects or levels approaching or reaching the levels in untreated normal subjects indicates a positive response to treatment.

[271] TTR amyloid levels can be measured by a number of methods, including imaging techniques. Examples of suitable imaging techniques include PET scanning with radiolabeled TTR of fragments thereof, TTR antibodies or fragments thereof, Congo-red-based amyloid imaging agents, such as, e.g., PIB (US 2011/0008255), amyloid-imaging peptide p31 (Biodistribution of amyloid-imaging peptide, p31, correlates with amyloid quantitation based on Congo red tissue staining, Wall etal, Abstract No. 1573, 2011 ISNM Annual Meeting), and other PET labels. Levels of non-native forms of TTR can be measured, for example, by performing SDS-PAGE/Western blot or Meso Scale Discovery plate assays with the antibodies disclosed herein on plasma samples or biopsy samples from a subject and comparing to control samples, as described in the examples.

A. Diagnostics and Monitoring Methods

1 Monitoring signs and/or symptoms

[272] The invention provides methods of monitoring of signs and/or symptoms of subjects having or at risk of TTR amyloidosis from related signs and/or symptoms. Some such subjects have been diagnosed with a TTR amyloidosis, e.g., by other methods described herein, such as from presence of TTR deposits, TTR level in blood, plasma or serum, or presence of a mutation in a gene encoding TTR. Such methods provide an indication of a subject’s conditions with or without concomitant treatment. The method can be used to control treatment. For example, the methods can be used to determine when to initiate treatment as signs and/or symptoms deteriorate relative to control value determined either in subjects free of TTR amyloidosis (negative controls) or subjects known to have TTR amyloidosis (positive controls). The methods can also be used to a monitor a subject’s response to treatment, for example, immunotherapy with an antibody specifically binding to TTR. The methods can also be used to modify treatment (e.g., change dose or frequency of existing treatment agent, or switch to a new treatment agent depending on the signs and/or symptoms). The methods can also be used to determine effectiveness of treatment and impact of treatment on a subject's quality of life. The methods can also be used to determine if treatment changes progression of disease, changes impact of symptoms on a subject, or changes a subject’s daily functioning.

[273] Examples of signs and/or symptoms that can be monitored include dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition. In some methods, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of these signs and/or symptoms are monitored.

[274] Other signs and/or symptoms that can be monitored include fatigue; shortness of breath; dizziness or fainting; chest pain; sleep disturbance; pain, numbness or tingling in feet or legs; pain, numbness or tingling in hands or arms; loss of sensitivity to temperature; swelling of legs or ankles; muscle weakness or loss of strength; pain; carpal tunnel syndrome; feeling full quickly when eating; nausea; vomiting; weight loss; diarrhea; constipation; urinary incontinence; blurred vision; sexual dysfunction including erectile dysfunction, vaginal pain or dryness, decreased libido, or other sexual dysfunction; blood in urine; spinal stenosis; stress; anxiety; depression and sensitivity to alcohol. Some methods monitor at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or all of these signs and/or symptoms.

[275] Monitoring can include determination of signs and/or symptoms on a single occasion or on multiple occasions, which may include, for example, determining a baseline value before initiating treatment. Monitoring can include determination(s) of signs and/or symptoms before and after initiating or modifying treatment. Modification of treatment can be performed once or multiple times in response to changing signs and/or symptoms. Monitoring of signs and/or symptoms can be performed at regular or irregular intervals. If regular intervals, the intervals can be e.g., weekly, monthly quarterly, every six months or every year. Monitoring can continue for e.g., at least a year, 5 years, 10 years or the life of a subject.

[276] Monitoring can be self-performed by a subject, or can be performed by an observer other than the subject, such as physician. Monitoring can be performed by a questionnaire such as provided in the examples. Monitoring can also involve recording of motion of a subject, such as with a video camera or smart phone app. Monitoring can also involve determining a score for cognitive testing.

[277] If two assessments of signs and/or symptoms are made, a direct comparison can be made between the two assessments to determine whether the signs and/or symptoms improved, deteriorated or remained the same between the two assessments. If more than two measurements are made, the measurements can be analyzed as a time course starting before immunotherapy and proceeding through immunotherapy. Signs and/or symptoms of a subject can also be compared with signs and/or symptoms of negative (i.e., healthy subjects) or positive (subjects with TTR amyloidosis) control populations. Comparative analysis of signs and/or symptoms can indicate whether signs and/or symptom(s) improved, deteriorated or remained the same in response to immunotherapy. Comparisons can be performed in a suitably programmed computer, which can also be programmed to provide output.

[278] Reference to an improvement or deterioration in signs and/or symptoms means an improvement which in the physician’s judgment is more likely than not due to the treatment rather than random variation in the subject’s condition, and is preferably demonstrated by an improvement beyond at least one and preferably two standard deviations of such fluctuation. In some methods, a value of an index representing a subject’s overall condition is calculated based on monitoring any combination or all of the above signs and/or symptoms. The index can weight a sign or symptom by the severity, impact on daily life and/or frequency of a sign or symptom in a subject and/or the sign or symptom itself. The value of an index can be compared with a mean value of the index in healthy subjects with no known presence or risk of TTR amyloidosis (i.e., healthy subjects). Such a value represents a negative control. The value of the index can also be compared with a mean value of the index in subjects known to have TTR amyloidosis (positive controls). For example, index scoring can be from 0 to 100 with lower scores indicating lower health status and quality of life, and higher scores indicating higher health status and quality of life. Changes in frequency and magnitude of symptoms from baseline scores are calculated to assess the impact and effectiveness of treatment on symptoms and impact on daily functioning as well as health related quality of life.

[279] Changes in a subject’s signs and/or symptoms, e.g., assessed from the value of the index in a subject relative prior value(s) in the subject or negative or positive controls, can be used in determining when to initiate treatment of a subject. For example, an index differs significantly (e.g., at least two standard deviations) from a mean in healthy subjects in a direction indicating increased signs and/or symptoms can provide an indication to initiate treatment. Likewise a value of an index in a subject approaching, at or exceeding a mean value in subjects with TTR amyloidosis can also provide an indication to initiate treatment. Changes in the index in the subject over time toward TTR amyloidosis can also provide an indication to initiate treatment.

[280] In some subjects receiving treatment for TTR amyloidosis, monitoring of treatment is used to indicate whether the treatment should be modified or continued as is. As in other methods, an index of signs and/or symptoms can be used to assess changes in signs and/or symptoms over time. Modification includes changing the dose or frequency of administration of an agent already being administered, and changing to a different agent. For example, monitoring indicating signs and/or symptoms are improving, remaining constant or deteriorating more slowly than expected provides an indication that an existing treatment should be continued either as is, or at reduced dose or frequency to determine if the same efficacy can be obtained for reduced drug administered. Dose or frequency can be varied by a factor of e.g., 1.5, 2, 3 or 5.

For example, if a subject is receiving an antibody specifically binding to TTR and the subject’s signs and/or symptoms improve, then the dosage can be titrated down to determine if the improvement continues at reduced dose with possible reduction in side effects from the reduced dose. If monitoring indicates signs and/or symptoms are deteriorating, particularly at a faster rate than is typical in untreated subjects, the monitoring provides an indication that the treatment should be modified either by increasing the dose or frequency of the existing agent or switching to a new agent. For example, if a subject is being treated with an agent other than an antibody against TTR and monitoring shows a deterioration in signs and/or symptoms, then the subject can be switched to treatment with an antibody against TTR. [281] The method can be for approved immunotherapeutic agents or as part of a clinical or preclinical trial of an immunotherapeutic agent. The methods can be practiced on a single individual or a population of individuals. If a population, then the population is preferably sufficiently large as to include at least one individual whose signs and/or symptoms decrease in response to treatment and at least one individual whose signs and/or symptoms remain the same or get worse after treatment. The population can be subjects treated by a particular physician or institution. The population can have at least, 2, 5, 10, 20, 50, 100, 500 or 1000 subjects.

[282] Also provided are methods of diagnosing TTR amyloidosis by monitoring signs and/or symptoms including dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition. In some methods, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of these signs and/or symptoms are monitored.

[283] Also provided are methods of diagnosing TTR amyloidosis by monitoring signs and/or symptoms including fatigue; shortness of breath; dizziness or fainting; chest pain; sleep disturbance; pain, numbness or tingling in feet or legs; pain, numbness or tingling in hands or arms; loss of sensitivity to temperature; swelling of legs or ankles; muscle weakness or loss of strength; pain; carpal tunnel syndrome; feeling full quickly when eating; nausea; vomiting; weight loss; diarrhea; constipation; urinary incontinence; blurred vision; sexual dysfunction including erectile dysfunction, vaginal pain or dryness, decreased libido, or other sexual dysfunction; blood in urine; spinal stenosis; stress; anxiety; depression and sensitivity to alcohol. Some methods monitor at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or all of these signs and/or symptoms.

[284] In some methods of diagnosing TTR amyloidosis, monitoring of signs and/or symptoms including dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition; and/or monitoring signs and/or symptoms including fatigue; shortness of breath; dizziness or fainting; chest pain; sleep disturbance; pain, numbness or tingling in feet or legs; pain, numbness or tingling in hands or arms; loss of sensitivity to temperature; swelling of legs or ankles; muscle weakness or loss of strength; pain; carpal tunnel syndrome; feeling full quickly when eating; nausea; vomiting; weight loss; diarrhea; constipation; urinary incontinence; blurred vision; sexual dysfunction including erectile dysfunction, vaginal pain or dryness, decreased libido, or other sexual dysfunction; blood in urine; spinal stenosis; stress; anxiety; depression and sensitivity to alcohol, is performed in addition to assessments including imaging such as electrocardiogram (ECG or EKG) to assess amyloid deposits in the heart or MRI to provide tissue characterization. Other diagnostic tools include blood tests, biopsies, or genetic testing.

[285] In some diagnostic methods, a value of an index representing a subject’s overall condition is calculated based on monitoring any combination or all of the above signs and/or symptoms. The index can weight a sign or symptom by the severity, impact on daily life and/or frequency of a sign or symptom in a subject and/or the sign or symptom itself. The value of an index can be compared with a mean value of the index in healthy subjects with no known presence or risk of TTR amyloidosis (i.e., healthy subjects). Such a value represents a negative control. The value of the index can also be compared with a mean value of the index in subjects known to have TTR amyloidosis (positive controls).

[286] Diagnosis can be based symptoms alone or a combination of symptoms and biochemical signs of TTR amyloidosis, such as presence of TTR deposits, altered TTR level in blood, plasma or serum from healthy subjects, or presence of a mutation in a gene encoding TTR. For example, a subject identified as having presence of TTR deposits, altered TTR level in blood, plasma or serum from healthy subjects, or presence of a mutation in a gene encoding TTR can be diagnosed as having TTR amyloidosis on a further finding of one or more of the symptoms listed above, or an index value differing from a mean value in healthy subjects. Alternatively, an individual presenting with one or more symptoms listed above, or an index value differing from a mean value in health subjects can be subject to imaging for TTR deposits, lab tests to determine TTR level in blood, plasma or serum or genetic testing to look for mutations in a TTR gene. Monitoring any combination or all of the above signs and/or symptoms can be used in combination with clinical assessments to incorporate a patient’s perspective in confirming symptoms in a quantitative manner. A patient who has broad symptoms involving multiple organs can be confirmed as having ATTR amyloidosis alongside biopsies, genetic testing, and/or imaging such as electrocardiogram (ECG or EKG) to assess amyloid deposits in the heart or MRI to provide tissue characterization.

2 Detecting Immune Response against TTR

[287] Also provided are methods of detecting an immune response against TTR in a subject suffering from or susceptible to diseases associated with TTR deposition or pathogenic forms of TTR ( e.g ., monomeric, misfolded, aggregated, or fibril forms of TTR). The methods can be used to monitor a course of therapeutic and prophylactic treatment with the agents provided herein. The antibody profile following passive immunization typically shows an immediate peak in antibody concentration followed by an exponential decay. Without a further dose, the decay approaches pretreatment levels within a period of days to months depending on the half-life of the antibody administered. For example, the half-life of some human antibodies is of the order of 20 days.

[288] In some methods, a baseline measurement of antibody to TTR in the subject is made before administration, a second measurement is made soon thereafter to determine the peak antibody level, and one or more further measurements are made at intervals to monitor decay of antibody levels. When the level of antibody has declined to baseline or a predetermined percentage of the peak less baseline (e.g., 50%, 25% or 10%), administration of a further dose of antibody is administered. In some methods, peak or subsequent measured levels less background are compared with reference levels previously determined to constitute a beneficial prophylactic or therapeutic treatment regimen in other subjects. If the measured antibody level is significantly less than a reference level (e.g, less than the mean minus one or, preferably, two standard deviations of the reference value in a population of subjects benefiting from treatment) administration of an additional dose of antibody is indicated.

3 Detecting misfolded TTR

[289] Also provided are methods of detecting monomeric, misfolded, aggregated, or fibril forms of TTR in a subject, for example, by measuring TTR amyloid or pathogenic forms of TTR (e.g, monomeric, misfolded, aggregated, or fibril forms of TTR) in a sample from a subject or by in vivo imaging of TTR in a subject. Such methods are useful to diagnose or confirm diagnosis of diseases associated with such pathogenic forms of TTR ( e.g ., TTR amyloidosis), or susceptibility thereto. The methods can also be used on asymptomatic subjects. The presence of monomeric, misfolded, aggregated, or fibril forms of TTR indicates susceptibility to future symptomatic disease. The methods are also useful for monitoring disease progression and/or response to treatment in subjects who have been previously diagnosed with a TTR amyloidosis.

[290] Biological samples obtained from a subject having, suspected of having, or at risk of having a TTR amyloidosis can be contacted with the antibodies disclosed herein to assess the presence of monomeric, misfolded, aggregated, or fibril forms of TTR. For example, levels of monomeric, misfolded, aggregated, or fibril forms of TTR in such subjects may be compared to those present in healthy subjects. Alternatively, levels of TTR amyloid or pathogenic forms of TTR (e.g., monomeric, misfolded, aggregated, or fibril forms of TTR) in such subjects receiving treatment for the disease may be compared to those of subjects who have not been treated for a TTR amyloidosis. Some such tests involve a biopsy of tissue obtained from such subjects. ELISA assays can also be used, for example, for assessing levels of monomeric, misfolded, aggregated, or fibril forms of TTR in fluid samples. Some such ELISA assays involve anti -TTR antibodies that preferentially bind monomeric, misfolded, aggregated, or fibril forms of TTR relative to native tetrameric form of TTR.

[291] Some such tests are sandwich immunoassays. Some such immunoassays employ the Meso Scale Discovery (MSD) electrochemiluminescence platform (Meso Scale Diagnostics, Rockville, MD.) Some such immunoassays use electrochemiluminescent labels on reporter antibodies, e.g., MSD Assays (Meso Scale Diagnostics, Rockville, MD.) For example, the reporter antibody can be labeled with a SULFO-TAG label ((Meso Scale Diagnostics, Rockville, MD). Plates useful in electrochemiluminescent assays may incorporate electrodes (e.g., MSD plates (Meso Scale Diagnostics, Rockville, MD). Plates useful in electrochemiluminescent assays may incorporate electrodes in the bottom of each well (e.g., MSD plates, (Meso Scale Diagnostics, Rockville, MD). Some assays employ a labeled capture antibody. For example, the labeled capture antibody can be 9D5 or 18C5 or a humanized, chimeric, or veneered variant thereof. Some assays employ a labeled reporter antibody. For example, the labeled reporter antibody can be 9D5 or 18C5 or a humanized, chimeric, or veneered variant thereof. The labeled reporter antibody can also be an antibody of Table 3, or a humanized, chimeric, or veneered variant thereof. The labeled reporter antibody can be an antibody that binds TTR with no conformational specificity. In an embodiment, the antibody that binds TTR with no conformational specificity can be 8C3 or 7G7 or a humanized, chimeric, or veneered variant thereof (See, e.g., WO 2016/120811). In an embodiment, the antibody that binds TTR with no conformational specificity can be a polyclonal antibody. In an embodiment, the polyclonal antibody is a polyclonal rabbit anti-human prealbumin (Cat. No. A000202-2, Dako, Agilent Technologies, Inc, Santa Clara, CA). In an embodiment, the polyclonal rabbit anti-TTR antibody is Sigma, Catalog No. HPA002550 (Sigma-Aldrich, St. Louis, MO),

[292] Some assays detect all misfolded TTR in a sample (i.e., all misfolded forms of TTR including monomers and multimers). Other assays specifically detect monomeric misfolded TTR or multimeric misfolded TTR. Other assays detect all forms of TTR (misfolded forms and native tetrameric form). Some such assays employ a capture antibody that specifically binds to an epitope within residues 89-97 of TTR or an epitope within residues 101-109 of TTR and a reporter antibody that specifically binds to a different epitope of TTR; wherein if misfolded TTR is present in the sample, the capture antibody and reporter antibody bind to the misfolded TTR forming a sandwich complex; and wherein detection of the reporter antibody that binds to the misfolded TTR, if any, indicates presence or absence of all the misfolded forms of TTR present in the sample. Such reporter antibodies can include 18C5, 9D5, 14G8, 5A1, 6C1, AD7F6, RT24, NI-301.35G11, MFD101, MDF102, MFD103, MFD105, MFD107, MFD108, MFD109,

MFDl 11, MFD 114, or a chimeric version or humanized version thereof. Such reporter antibodies can include an antibody which binds within residues 89-97, 101-109, 118-122, 115- 124, 53-63, 54-61, 36-49, 49-61, 109-121, 30-66, 70-127, 80-127, 90-127, 100-127, 110-127, or 115-127 of TTR. Such reporter antibodies can include 8C3 or 7G7 (see, e.g., WO 2016/120811). Such reporter antibodies can include a polyclonal rabbit anti-human prealbumin (Cat. No. A000202-2, Dako, Agilent Technologies, Inc, Santa Clara, CA) or a polyclonal rabbit anti-TTR antibody (Sigma, Catalog No. HPA002550, Sigma-Aldrich, St. Louis, MO). Some such assays detect misfolded forms of TTR in a biological sample from hereditary TTR amyloidosis subjects carrying a mutation at position 89 within the 9D5 epitope of TTR. Exemplary mutations are E89K TTR and E89Q TTR. Some such assays employ a 9D5 capture antibody and a polyclonal anti-TTR reporter antibody or an 18C5 reporter antibody. [293] Some assays detect multimeric forms of misfolded TTR in a sample. Such assays can be configured to detect multimeric misfolded TTR preferentially or exclusively over monomeric misfolded TTR. Some such assays employ a capture antibody that specifically binds to an epitope within residues 89-97 or 101-109 of TTR and a reporter antibody that specifically binds to an epitope within residues 89-97 or 101-109 of TTR. Such a combination of capture and reporter antibodies can bind preferentially or exclusively to multimeric TTR over monomeric because the multiple copies of TTR provide multiple epitopes for antibody binding. Detection of reporter antibody binding to multimeric misfolded TTR, if any, indicates presence or absence of the multimeric misfolded TTR. In some such assays, the reporter antibody competes for binding TTR with the capture antibody and/or the reporter and capture antibody bind to the same or overlapping epitope of TTR. In some such assays the capture antibody binds a first misfolded TTR molecule in the multimeric misfolded TTR, and the reporter antibody binds a second misfolded TTR molecule in the multimeric misfolded TTR. Competition for binding between the capture and the reporter antibodies precludes or at least reduces (depending on whether competition is the result of overlapping epitopes or steric hindrance) simultaneous binding and detection of monomeric misfolded TTR. In some such assays, detection of the reporter antibody binding that binds to the second misfolded TTR molecule in the multimeric TTR indicates presence or absence of multimeric misfolded TTR.

[294] The antibodies disclosed herein can be used in a method of determining a ratio of the level of total multimeric misfolded transthyretin (TTR) to the level of total misfolded TTR in a biological sample. A first portion of a biological sample can be assayed for all misfolded TTR in a sample (i.e., all misfolded forms of TTR including monomers and multimers) in a first assay wherein monomeric misfolded and multimeric misfolded TTR are detected. The first assay can employ a capture antibody that specifically binds to an epitope within residues 89-97 or 101-109 of TTR and a reporter antibody that specifically binds to a different epitope of TTR. If misfolded TTR is present in the sample, the capture antibody and reporter antibody bind to the misfolded TTR forming a sandwich complex. Detection of the reporter antibody that binds to the misfolded TTR, if any, indicates presence or absence of the misfolded TTR in the sample. A second portion of a biological sample can be assayed for multimeric forms of misfolded TTR a biological sample in a second assay that detects multimeric misfolded TTR preferentially over monomeric misfolded TTR. The second assay can employ a capture antibody that specifically binds to an epitope within residues 89-97 or 101-109 of TTR and a reporter antibody that specifically binds to an epitope within residues 89-97 or 101-109 of TTR. If multimeric misfolded TTR is present in the sample, the capture antibody and reporter antibody bind to the multimeric misfolded TTR forming a sandwich complex. The capture and the reporter antibody can bind simultaneously preferentially or exclusively to the multimeric misfolded TTR, if any, to indicate presence or absence of the multimeric misfolded TTR. In some such assays, the reporter antibody competes for binding TTR with the capture antibody or binds to the same or overlapping epitope as the capture antibody. In some such assays the capture antibody binds a first misfolded TTR molecule in the multimeric misfolded TTR, and the reporter antibody binds a second misfolded TTR molecule in the multimeric misfolded TTR. Competition for binding between the capture and the reporter antibodies precludes or at least reduces (depending on whether competition is the result of overlapping epitopes or steric hindrance) simultaneous binding and detection of monomeric misfolded TTR. In some such assays, detection of the reporter antibody binding that binds to the second misfolded TTR molecule in the multimeric TTR indicates presence or absence of multimeric misfolded TTR. In some assays, a ratio of multimeric misfolded TTR to all misfolded TTR is calculated.

[295] The antibodies disclosed herein can also be used in a method of determining a ratio of the level of all misfolded TTR to total TTR (misfolded forms and native tetrameric form) in a biological sample. A first portion of a biological sample can be assayed for all misfolded TTR in a sample (i.e., all misfolded forms of TTR including monomers and multimers), in a first assay wherein monomeric misfolded and multimeric misfolded TTR are detected. The first assay can employ a capture antibody that specifically binds to an epitope within residues 89-97 or 101-109 of TTR and a reporter antibody that specifically binds to a different epitope of TTR. If misfolded TTR is present in the sample, the capture antibody and reporter antibody bind to the misfolded TTR forming a sandwich complex. Detection of the reporter antibody binding to the misfolded TTR, if any, indicates presence or absence of the misfolded TTR in the sample. A second portion of a biological sample can be assayed for total TTR (misfolded forms and native tetrameric form) in a second assay wherein total TTR is detected. The second assay can employ a capture antibody that binds TTR with no conformational specificity and a reporter antibody that binds TTR with no conformational specificity. If TTR is present in the sample, the capture antibody and reporter antibody bind to the TTR forming a sandwich complex. Detection of the reporter antibody binding to the TTR, if any, indicates presence or absence of TTR present in the sample. A ratio of all misfolded TTR to total TTR (misfolded forms and native tetrameric form) can be calculated.

[296] The in vivo imaging methods can work by administering a reagent, such as antibody that binds to monomeric, misfolded, aggregated, or fibril forms of TTR in the subject, and then detecting the reagent after it has bound. Such antibodies typically bind to an epitope within residues 89-97 or 101-109 of TTR. If desired, the clearing response can be avoided by using antibody fragments lacking a full length constant region, such as Fabs. In some methods, the same antibody can serve as both a treatment and diagnostic reagent.

[297] Diagnostic reagents can be administered by intravenous injection into the body of the subject, or via other routes deemed reasonable. The dose of reagent should be within the same ranges as for treatment methods. Typically, the reagent is labeled, although in some methods, the primary reagent with affinity for monomeric, misfolded, aggregated, or fibril forms of TTR is unlabeled and a secondary labeling agent is used to bind to the primary reagent. The choice of label depends on the means of detection. For example, a fluorescent label is suitable for optical detection. Use of paramagnetic labels is suitable for tomographic detection without surgical intervention. Radioactive labels can also be detected using PET or SPECT.

[298] Diagnosis is performed by comparing the number, size, and/or intensity of labeled loci to corresponding base line values. The base line values can represent the mean levels in a population of undiseased individuals. Base line values can also represent previous levels determined in the same subject. For example, base line values can be determined in a subject before beginning treatment, and measured values thereafter compared with the base line values.

A decrease in values relative to base line generally signals a positive response to treatment.

[299] Monitoring of changes in amount of misfolded TTR, multimeric misfolded TTR, transthyretin deposits, anti-TTR antibody binding or the like allows adjustment of a treatment regimen in response to the treatment. Then a determination can be made whether to adjust treatment and if desired treatment can be adjusted in response to the monitoring. A significant change means that comparison of the value of a parameter after treatment relative to basement provides some evidence that treatment has or has not resulted in a beneficial effect. In some instances, a change of values of a parameter in a subject itself provides evidence that treatment has or has not resulted in a beneficial effect. In other instances, the change of values, if any, in a subject, is compared with the change of values, if any, in a representative control population of subjects not undergoing treatment. A difference in response in a particular subject from the normal response in the control subject (e.g., mean plus variance of a standard deviation) can also provide evidence that a treatment regimen is or is not achieving a beneficial effect in a subject. Changes in the above TTR parameters can also be combined with other change(s) in signs or signs and/or symptoms such as side effects in determining whether and how to adjust treatment.

[300] In some subjects, monitoring indicates that the amount of misfolded TTR, multimeric misfolded TTR, transthyretin deposits, or anti-TTR antibody binding is the same or greater than previously detected. In such subjects, if there are no unacceptable side effects, the treatment regimen can be continued as is or even increased in frequency of administration and/or dose if not already at the maximum recommended dose.

[301] In some subjects, monitoring indicates a detectable decline in amount of misfolded TTR, misfolded multimeric TTR, transthyretin deposits, anti-TTR antibody binding or the like but that amount of misfolded TTR, multimeric misfolded TTR, transthyretin deposits, or anti-TTR antibody binding remains above normal. In such subjects, if there are no unacceptable side effects, the treatment regimen can be continued as is or even increased in frequency of administration and/or dose if not already at the maximum recommended dose. Alternatively, in some such subjects, the treatment regimen can be discontinued and replaced with treatment with other agents, such as a TTR tetramer stabilizer, an antisense oligonucleotide based therapeutic, an RNA interference (RNAi) based therapeutic or doxycycline plus tauroursodeoxycholic acid.

[302] If the monitoring indicates an amount of misfolded TTR, multimeric misfolded TTR, transthyretin deposits, anti-TTR antibody binding or the like in a subject has already been reduced to at or near a normal level of amount of misfolded TTR, multimeric misfolded TTR, transthyretin deposits, or amount of anti-TTR antibody binding, the treatment regimen can be adjusted from one of induction (i.e., that reduces the level of amount of misfolded TTR, multimeric misfolded TTR, transthyretin deposits, or anti-TTR antibody binding) to one of maintenance (i.e., that maintains amount of misfolded TTR, multimeric misfolded TTR, transthyretin deposits, or anti-TTR antibody binding at an approximately constant level). Such a regimen can be effected by reducing the dose and or frequency of administering the treatment. Alternatively, in some such subjects, the treatment regimen can be discontinued and replaced with treatment with other agents, such as a TTR tetramer stabilizer, an antisense oligonucleotide based therapeutic, an RNA interference (RNAi) based therapeutic or doxycycline plus tauroursodeoxycholic acid.

[303] In other subjects, monitoring can indicate that the treatment regimen is having some beneficial effect but a suboptimal effect. An optimal effect can be defined as a percentage reduction in amount of misfolded TTR, multimeric misfolded TTR, transthyretin deposits, or anti-TTR antibody binding within the top half or quartile of the change in amount of misfolded TTR, multimeric misfolded TTR, transthyretin deposits, or amount of anti-TTR antibody binding) experienced by a representative sample of subjects undergoing the treatment regimen at a given time point after commencing therapy. A subject experiencing a smaller decline or a subject whose amount of misfolded TTR, multimeric misfolded TTR, transthyretin deposits, or anti-TTR antibody binding remains constant or even increases but to a lesser extent than expected in the absence the treatment regimen (e.g., as inferred from a control group of subjects not administered the treatment regimen) can be classified as experiencing a positive but suboptimal response. Such subjects can optionally be subject to an adjustment of regimen in which the dose and or frequency of administration of an agent is increased. Alternatively, or additionally if upward adjustment does not result in an improved response, in some such subjects, the treatment regimen can be discontinued and replaced with treatment with other agents, such as a TTR tetramer stabilizer, an antisense oligonucleotide based therapeutic, an RNA interference (RNAi) based therapeutic or doxycycline plus tauroursodeoxycholic acid.

[304] In some subjects, amount of misfolded TTR, multimeric misfolded TTR, transthyretin deposits, or anti-TTR antibody binding may increase in similar or greater fashion to misfolded TTR, multimeric misfolded TTR, transthyretin deposits, or anti-TTR antibody binding in subjects not receiving treatment. If such increases persist over a period of time, treatment can if desired be discontinued in favor of treatment with one or more other agents. [305] Diagnostic methods with antibodies disclosed herein can be performed in combination with a second anti-TTR antibody that binds an epitope different from that of 9D5 or 18C5, for example an antibody as disclosed in Table 3.

[306] The assays disclosed herein can also be used to assess target engagement (pharmacodynamics effects) of unbound (free) misfolded-TTR in a biological sample from a subject by an antibody being used or tested for use in treatment. Such an antibody is referred to in the present assay as being a test antibody because it is being tested for its target engagement.

In the present assay, the biological sample can be an aliquot of a larger sample, referred to as a collected sample, such that the assay can be run in a parallel manner of multiple aliquots of the collected sample. The test antibody competes with the capture antibody (or alternatively the reporter antibody) for binding to TTR. The test antibody can bind to the same epitope as the capture antibody (or the reporter antibody). The sandwich assay described above can be run in parallel for first and second aliquots of a collected sample, which are usually of the same volume. One aliquot is supplemented with the test antibody and both aliquots are supplemented with capture and reporter antibodies. In the aliquot without the test antibody, detection of the reporter antibody as part of a sandwich provides an indication of presence and amount of misfolded TTR in a sample. In the aliquot with the test antibody, detection of a reduced amount of reporter antibody as part of a sandwich relative to the aliquot without the test antibody provides an indication the test antibody is binding to misfolded TTR and thereby competing with either the capture or reporter antibody and reducing formation of a sandwich between the capture antibody, misfolded TTR and the reporter antibody. Such an assay can be performed on additional aliquots containing increasing amounts of the test antibody (as well as capture antibody) further to characterize binding of the test antibody to misfolded TTR. The sample can be from a subject with a TTR amyloidosis. The sample can be from a subject with a hereditary TTR amyloidosis. The subject with a hereditary TTR amyloidosis may carry a mutation selected from the group consisting of V30M, Y114C, G47R, S50I, E61L, T49S, F33V, A45T, E89K, E89Q, and V122I. In some such assays, the biological sample is a plasma sample. In some such assays, the assay is performed with a 9D5 or 18C5 capture antibody and a polyclonal anti-TTR reporter antibody. Such an assay can be used to inform on target engagement in clinical trials of an antibody intended for therapeutic use as disclosed herein. In some such assays, the test antibody is 14G8, and the assay is performed with a 9D5 or 18C5 capture antibody and a polyclonal anti-TTR reporter antibody.

[307] The assays disclosed herein can be used to measure pharmocodynamic effects of therapies targeting misfolded forms of TTR. In some such assays, target engagement of unbound (free) mis-TTR in a biological sample is measured after ex-vivo treatment (spiking) of a biological sample with a test antibody. The assays disclosed herein can also be used to measure efficacy of a test antibody in a subject. Biological samples are collected from a subject before and after treatment with a test antibody. In some such assays, target engagement of unbound (free) mis-TTR in a biological sample is measured before and after in vivo treatment of a subject with a test antibody. In some assays the target of the test antibody is an epitope within residues 89-97 of TTR. In some assays the target of the test antibody is an epitope within residues 101- 109 of TTR.

[308] The present methods also allow distinction of a transthyretin amyloidosis from a non- TTR amyloidosis, e.g. amyloid light-chain (AL) amyloidosis, also known as primary systemic amyloidosis.

[309] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. EXAMPLES

[310] Example 1: Initial Survey Development

Study Goals

[311] The goals of this study were to evaluate the content validity of the ATTR amyloidosis symptom survey (ATTR-PSS) using a subject focus group in which the moderator employed both concept elicitation and cognitive debriefing techniques.

[312] The concept elicitation portion of the focus group was designed to confirm the findings of previous research efforts (ie, a brief literature review; discussions from a subject advisory board and experts in the field) and identify any additional concepts that should be added to the ATTR- PSS. Specifically, the goal was to identify:

1. The full range of symptoms experienced due to ATTR amyloidosis

2. Which symptoms impact subjects’ ability to function and perform activities of daily living

3. Which symptoms are the most burdensome?

4. How burdensome each symptom is, and in which ways?

5. Which symptoms, if any, only occur within subsets of individuals with shared disease characteristics; in particular, for subjects with polyneuropathy only, those with cardiomyopathy only, and those with both polyneuropathy and cardiomyopathy

[313] The primary goal of the cognitive debriefing portion of the focus group was to determine whether a recently developed ATTR amyloidosis symptom survey (ATTR-PSS) is relevant, comprehensive, and easy for subjects to understand and complete.

Methods

Focus Group Population and Recruitment

[314] Seven subjects were recruited from the Amyloidosis Research Consortium (ARC; a patient advocacy group) Efforts were made to recruit a sample of patients with varied representation in terms of age, gender, U.S. geographic region, type of ATTR amyloidosis, and treatment experiences. Interested individuals contacted ARC and were subsequently sent the informed consent form (ICF) to review in advance of the focus group. The study was approved by the New England Independent Review Board (NEIRB).

Inclusion/Exclusion Criteria

[315] Patients were eligible for the study if they:

1) were at least 18 years of age

2) self-reported having ATTR amyloidosis (as diagnosed by a doctor)

3) were willing and able to participate in-person in the study and comply with all study requirements including giving written informed consent.

Study Procedures

[316] Meeting rooms were equipped with audio recording equipment and one-way mirrors. Prior to commencing the focus group, upon arriving at the facility, patients individually completed the ICF. The first part of the focus group, the concept elicitation (CE) segment, followed a semi-structured format . First, the moderator and assistant prompted patients to verbally call out the different symptoms they had experienced due to their ATTR amyloidosis (referred to later as “verbalized symptoms”). After all of the patients had the opportunity to verbalize their symptoms, patients were asked to complete a symptom worksheet. Patients were provided with a list of 29 symptoms (not including “Other) and were instructed to place a check mark next to the symptoms that affected them, and then to rank-order their symptoms based on the impact each symptom had on their daily lives; lower scores indicated greater impact. In addition to the 29 symptoms included on the worksheet, there was a free-text option so that patients could report symptoms that were not included on the list. After completing the symptom checklist, the moderator led patients in a discussion regarding the ways in which various symptoms impacted their lives, and the frequency with which patients experience different symptoms.

[317] During the second segment of the focus group, the moderator conducted the cognitive debriefing (CD) of the ATTR-PSS. This segment followed a structured format in which the moderator asked the patients to review each element of the survey (ie, the overall format, title, instructions, items, recall period, and response choices) and to report whether each element was relevant, clear, and easily answerable. Patients were also encouraged to suggest any changes or alternative wording that might increase clarity.

Data Coding and Analysis

[318] The focus group transcript was coded and analyzed by an experienced qualitative researcher using an Excel database developed specifically for this study. Focus group content for the CE portion of the focus group was analyzed using grounded theory methods. Grounded theory is an inductive methodology in which the researcher allows concepts or themes to emerge from the transcript content rather than applying an a priori hypothesis regarding which concepts or themes should be present. The data from the CE portion of the focus group were analyzed for types of symptoms, prevalence and severity of symptoms, and areas of life impacted by ATTR amyloidosis.

[319] The CD portion of the focus group used a systematic, structured approach to evaluating the comprehensibility of the ATTR-PSS. Data from the CD portion of the focus group were analyzed to identify areas in which there was confusion or disagreement with elements of the ATTR-PSS using a pre-set, relevant threshold of agreement of (>25%). If 25% or more of patients in the focus group recommended the same change, the change was carefully reviewed within the study team and a recommendation was made to either make the change or document why the change was not needed.

Results

[320] Demographic and Disease Characteristics

[321] Table 4 contains the demographics and disease characteristics of the seven focus group patients:

[322] Table 4. Patient Characteristics

Patients could select more than one response. As such, percentages add to more than 100; treatment referred to any treatment ever received to treat ATTR amyloidosis specifically

Concept Elicitation Findings Symptoms of ATTR Amyloidosis Verbalized Symptoms

[323] Patients reported experiencing a variety of symptoms associated with ATTR amyloidosis. When asked to share their symptoms, patients spontaneously reported over 20 unique symptoms. Some symptoms had not previously been reported. These new symptoms are included in the questionnaire below:

• Loss of appetite

• Gas, bloating, urgency to eliminate

• Dementia

• Cognition/memory issues

• Seizures

• Stroke

• Sleep apnea [324] Sleep apnea emerged as a symptom in a later part of the CE phase in which patients discussed the ways in which their disease impacted their lives.

Symptom Worksheet: Prevalence and Imyact/Burden

[325] After listing out their symptoms spontaneously, patients filled out the symptom worksheet which allowed them to indicate which of the presented symptoms they had experienced, and then rank the symptoms in terms of how much they impact their life. All symptoms on the worksheet were endorsed by at least one patient, with the exception of “renal dysfunction.” Patients also used the open text field to write in and rank symptoms that did not already appear on the worksheet. While some of these handwritten symptoms had also been mentioned earlier during the first part of the concept elicitation phase (and are described in section 4.2.1.1), new symptoms were also reported, four of which were subsequently added to the revised survey:

[326] Dry eyes, dry mouth, severe headache and migraine, muscle cramps.

[327] A figure illustrating the symptoms experienced by least 4 of the 7 patients (57%) is provided in Figure 1 below. As can be seen in the figure, the two most commonly experienced symptoms were fatigue, and pain, numbness, or tingling in hands or arms; both of which were experienced by 6 patients.

[328] In examining symptoms that patients rated as being the most bothersome (ranked #1 on the worksheet), 4 of the 7 patients (57%) indicated that shortness of breath only during exercise or exertion was the most bothersome (note that all patients who reported experiencing this symptom also ranked it as the most bothersome). The remaining patients endorsed either fatigue; pain, numbness, or tingling in hands/arms; shortness of breath all the time; or anxiety as the most bothersome symptom (one patient endorsed 2 symptoms as the most bothersome). In examining symptoms that patients rated as being either the second or third most bothersome, 5 of 7 patients (71%) endorsed fatigue, while 3 of 7 (42.8%) endorsed pain, numbness, or tingling in feet or legs. The prevalence of patients who reported symptoms as being either the most burdensome or the second or third most burdensome, is illustrated in Figure 2. [329] Figure 1. Most prevalent symptoms reported on worksheet: Symptoms experienced by at least 4 of 7 patients

[330] Figure 2. Prevalence of symptoms according to amount of burden/impact Impacts of A TTR Amyloidosis

[331] Areas of impact were described by patients throughout various parts of the concept elicitation phase, including during the introductory phase, the symptom description phase, and during the guided discussion. Three areas of impact that emerged from the data are summarized below.

Overall Symptom Impact

[332] In addition to listing symptoms they experienced, patients also described their symptoms in ways that illustrated the impact these symptoms had on everyday life.

Impact on Family

[333] Patients indicated that amyloidosis had impacted their home and family life. All patients reported having a spouse and many specified that their partners were indispensable to navigating the physical limitations imposed by the disease. The toll that the disease took on family members appeared to be two-fold: first, there was the extra burden of work that fell on the non-amyloid spouse in managing the chores that the patient could no longer tend to.

[334] Second, spouses also experienced fear that their loved one’s health would deteriorate and/or that he or she was at imminent risk of death. Patients also reported guilt associated with the possibility of genetically transmitting the condition to their child.

Impact on Job

[335] Most patients were working at least part time, and multiple patients indicated that their symptoms posed challenges in the workplace. Another patient had a supervisory role within his company, and expressed reluctance to have his colleagues learn of his diagnosis for fear that it would change their perception of him within the work environment. Patients discussed their experiences contending with work responsibilities while dealing with the symptoms of their ATTR. Cognitive Debriefing Findings and Recommendations

Summary of findings

[336] Overall, patients reported that that they understood and agreed with the content of the survey. There was a great deal of discussion regarding preferences for questionnaire content, wording and recall period, but overall, changes to the survey were minimal. These changes included the removal of one symptom (renal dysfunction), edits to three other symptoms (two related to breathing, one related to pain), and revisions to improve the clarity of response options. Each of the relevant parts of the survey is discussed in more detail in the following sub-sections.

Symptom list

[337] The first three items of the survey include a list of 29 symptoms for patients to evaluate. As discussed in previous sections, patients had the opportunity to record which of these symptoms they have experienced. During the cognitive debriefing phase of the focus group, patients discussed their experience with, and understanding of the symptoms. Symptoms that were discussed as being confusing or potentially problematic are described in additional detail below.

Renal Dysfunction

[338] Consistent with the finding from the concept elicitation phase, in which no patients reported experiencing renal dysfunction, a patient expressed confusion regarding the exact nature of the symptom and wondered how she would know if she had it:

P4: Like how do you know if you have renal dysfunction? Are you supposed to remember the report from your — Is that the kidney?... And how do I know that I have that?

[339] Because issues arose with this symptom during both phases of the focus group (lack of symptom experience and confusion regarding symptom), Optum omitted this symptom from the revised survey.

Shortness of Breath [340] As described in section 4.2.1.2, the majority of patients reported having experienced both shortness of breath all the time (5 of 7 patients, 71%) and shortness of breath only during exercise (5 of 7 patients, 71%). When given the opportunity to discuss the symptoms in more detail, multiple patients expressed confusion about the distinction between these two symptoms. Based on this feedback, the two different symptoms were replaced with one symptom, “Shortness of breath.”

Pain

[341] Throughout the course of the focus group, the experience of pain was mentioned by multiple patients. While reviewing the ATTR-PSS, one patient put a question mark next to the symptom “Pain, not related to neuropathy,” potentially indicating a source of confusion. It was decided to shorten the symptom description to “pain,” to potentially alleviate confusion.

Item Text and Response Options

[342] Each of the four items can be divided into two sections: the item text and the response options. Overall, patients reported no difficulty in comprehending the item text, though a small number patient offered suggestions. Additional suggestions were provided to improve the response options, as described below.

Item 1 Endorsement of Symptoms Item Text

[343] Item 1 asks patients, “In the past month, how often have you experienced each of the following symptoms?” There were no reported issues with this item’s wording or clarity.”

Response Options

[344] To respond to Item 1, patients were asked to comment on the frequency with which they experienced the 29 listed symptoms using a five point response scale ranging from “Never” to “All of the time or almost all of the time.”

[345] Based on this feedback, and because it represented a clear potential source of confusion to patients, the response choices were revised such that “Often” was changed to “Most of the time” and “Almost all of the time” was removed from the phrase used in the final response option..

Item 2 Severity of Symptoms

Item Text

[346] Item 2 asks patients, “In the past month, how severe were each of the following symptoms you have experienced?” Some patients expressed preferences such as removing the word “severe” from the item, because it might bias patients to thinking about their symptoms being severe. However, because these suggestions indicated individual preferences rather than specific sources of confusion, no edits were made.

Response Options

[347] To respond to Item 2, patients were asked to comment on the severity of the 29 listed symptoms with a five point response scale ranging from “Not at all” to “Very severe.”

[348] Two patients indicated that they did not understand the difference between "Severe" and "Very severe."

[349] This feedback was noted, but the original response options were ultimately retained because the response options are standard for patient surveys and the belief that most patients would be able to differentiate between “Severe” and “Very severe” symptoms, as 5 of the 7 patients did.

Item 3 Impact of Symptoms

Item Text

[350] Item 3 asks patients, “Of all the symptoms you have experienced due to ATTR amyloidosis in the past month, which had the most significant impact on your daily life? A significant impact is when a symptom prevents you from functioning physically, emotionally, or socially. Please select up to five symptoms.” Patients provided feedback on two aspects of this item, specifically related to the phrase “significant impact” and the request to rank symptoms. [351] Two patients noted issues with the description of the phrase “significant impact,” which is defined in the second sentence of the item text.

[352] The text of this item was subsequently streamlined by removing the sentence that provided a definition of “significant impact.” The word “symptoms” was also reiterated in the first sentence of the item text.

[353] Two patients also questioned the restriction on selecting only five symptoms that have the greatest impact, as the ability to include more than five may help to more clearly characterize the patients’ symptom experience. However, after additional discussion, one of the patients ultimately decided that the limit might be useful to researchers, as it provides more focused information on the areas that may require the most attention

Response Options

[354] Because patients were asked to check boxes to indicate which five of the 29 symptoms had the greatest impact on their lives, there were no response options associated with this item, eliminating the potential for revision or modification of response options for this item.

Item 4 Overall Symptom Severity Item Text

[355] Item 4 asks patients, “Overall, how severe are your ATTR amyloidosis symptoms?” Because the majority of patients did not indicate agreement with this sentiment, nor did they note additional problems with this question, the original wording was retained.

Response Options

[356] In response to Item 4, patients were asked to check one of six response options ranging from “Not severe at all,” to “Extremely severe.”

[357] Overall, the feedback from the patients reflected the desire to simplify the response options to make them easier to understand. That said, there was no specific recommendation made by more than one patient, and there was a person who disagreed with the specific suggestions given and noted that the response options were fine as they were. [358] Based on this, Optum chose to reduce the scale from six to five options and, for greater consistency and clarity, used the same options that were presented in item 2.

Recall Period

[359] Items 1-3 of the ATTR-PSS ask patients to report on symptoms that they have experienced “in the past month.”

[360] As reflected in the quotes provided above, multiple patients made suggestions, but no consensus was ultimately reached regarding an optimal recall period or description. As such, Optum chose not to modify it..

Summary and Next Steps

[361] This phase of ATTR-PSS development involved a focus group composed of seven patients with different types of ATTR amyloidosis, with two primary goals:

1. Determine whether the survey adequately captures the range of symptoms, and the frequency, severity, and impact these symptoms have on individuals with ATTR amyloidosis through concept elicitation

2. Assess the ease, comprehensibility, and appropriateness of both the survey overall and the individual components of the survey (e.g. instructions, item text, response options, recall period, etc.) through cognitive debriefing

[362] Based on the responses and feedback provided from patients, a small set of changes were made to the survey:

• Addition of 12 symptoms

• Removal of one symptom (renal dysfunction)

• Combining of two symptoms into one single item (difficulty breathing),

• Reducing the description associated with one symptom (pain)

• Reducing text in item 3 that defined the concept “significant impact”

• Modifying the response options for items 2 and 4 [363] These changes resulted in a net addition of 10 symptoms, for a total of 39 (not including “Other”). Two other limitations are worth noting: (1) there were no patients with only cardiomyopathy, although there were patients with both cardiomyopathy and polyneuropathy; (2) inclusion criteria were broad and did not include any quotas to ensure representation across types of ATTR amyloidosis.

[364] ATTR Amyloidosis Patient Symptom Survey

ATTR Amyloidosis Patient Symptom Survey (ATTR-PSS)

This survey is about symptoms you may have experienced related to ATTR amyloidosis. For each symptom listed, please select the one response that best describes your answer.

Abbreviations

[365] Example 2: Content Validation of the ATTR Amyloidosis Patient Symptom Survey: Findings from patient and clinician cognitive debriefing interviews

[366] Objective: The ATTR Patient Symptom Survey (ATTR-PSS) was previously developed through literature review and concept elicitation input from clinicians and patients and revised after evaluation by a patient focus group. This study further evaluated the content validity of the ATTR-PSS through qualitative cognitive debriefing interviews with clinicians and patients.

[367] Methods: Seven clinicians and 10 patients with ATTR amyloidosis were interviewed individually regarding their overall impressions, the clarity and appropriateness of the survey, relevance of concepts measured, and comprehensiveness and comprehensibility of items and response choice sets.

[368] Results: Clinicians acknowledged the usefulness of the ATTR-PSS in research and clinical settings. They suggested minor modifications to the survey instructions, the addition of 3 symptoms, and the transfer of 10 conditions from the symptom list to 2 separate items. Patients found the ATTR-PSS to be easy to complete and relevant to their experiences. Their feedback resulted in modification to instruction text, edits to the description of 4 symptoms, removal of 1 symptom, and addition of 2 diagnoses.

[369] Conclusion: The findings support the content validity of the ATTR-PSS as an appropriate measure of symptom frequency, severity, and impact in patients with ATTR amyloidosis.

[370] Introduction: The ATTR Patient Symptom Survey (ATTR-PSS) is a patient recorded outcome (PRO) developed in 2017 to assess the type, frequency, severity, and degree of impact of symptoms experienced by patients with ATTR amyloidosis. The ATTR-PSS was designed to be applicable to patients with either ATTRm or ATTRwt. Establishing the content validity of an instrument is a necessary component of PRO measure development, as it provides evidence that the measure assesses content areas that are appropriate and comprehensive given the intended population, and that items are easily understood and accurately interpreted by respondents [U.S. Department of Health and Human Services, Food and Drug Administration. Guidance for industry patient-reported outcome measures: Use in medical product development to support labeling claims; 2009. ://www.fda.gov/regulatory-information/search-fda-guidance- documents/patient-reported-outcome-measures-use-medical -product-development-support- labeling-claims. Accessed July 11, 2019]

[371] ]The initial development of the ATTR-PSS was informed by 1) literature review, 2) initial content validation through informal review and discussion with clinicians, 3) concept elicitation input from a patient advisory board, 4) review by a patient advocacy group, and 5) a formal concept elicitation and cognitive debriefing with a patient focus group that resulted in minor revisions to the survey (Figure 3). While initial evidence from the patient focus group supported the content validity of the ATTR-PSS, the content validation efforts to-date had not included a formal review by clinicians and included only a small number of patients in 1 focus group. Thus, it was determined that additional research should be conducted. This example presents the findings of a series of cognitive debriefing interviews with both clinicians and patients, with the primary objective of providing additional evidence of the content validity of the ATTR-PSS.

Materials and Methods

[372] Two sets of cognitive debriefing interviews were conducted consecutively to evaluate the content validity of the ATTR-PSS: 1) interviews with clinicians who treat patients with ATTR amyloidosis; and 2) interviews with patients diagnosed with ATTR amyloidosis.

Participants

[373] Seven clinicians and 10 patients were recruited for the study. Clinicians with experience treating patients with ATTR amyloidosis were contacted via email. Efforts were made to invite both male and female clinicians who practiced in different geographic regions. Clinicians who responded to the email were scheduled for a telephone interview; each clinician was provided a toll-free number to use for the call and a copy of the ATTR-PSS prior to the interview.

[374] Patients were recruited for the study through collaboration with the Amyloidosis Support Groups (ASG); information about the study was distributed through ASG’s social media pages. Patients were eligible to participate if they were at least 18 years of age, reported having been diagnosed by a doctor with ATTR amyloidosis, and were comfortable reading and communicating in English. A quota system was developed to include representation of patients with different types of ATTR amyloidosis, to ensure that different symptom experiences were captured in the interviews. The quota was set to include at least 2 patients with each of the following types: ATTR-PN, ATTR-CM, ATTR-PN and CM, and ATTRwt. Because efforts were focused primarily on achieving diversity in ATTR amyloidosis type, and ATTR amyloidosis is a rare disease (making recruitment especially challenging), no formal quotas were implemented for other patient characteristics such as age, gender, education, or time since diagnosis. ATTR-PSS

[375] The draft version of the ATTR-PSS included a list of 40 different symptoms experienced by patients who have ATTR amyloidosis. In reference to the symptom list, patients are asked to indicate 1) how often they have experienced each symptom; 2) the severity of each symptom; 3) the 5 symptoms that have had the greatest impact on their daily life; and 4) the overall severity of their symptoms. All 4 items include a recall period of ‘the past month.’

Study procedures

[376] The clinician interviews were conducted by phone in March 2019. Approximately 1 week prior to the interview, clinicians were emailed a copy of the ATTR-PSS to review. At the start of the interview, the interviewer provided a brief description of the purpose of the interview and obtained permission from the clinician to audio-record the interview. The interviews followed a semi-structured interview guide; the topics included in the guide are depicted in Figure 4.

[377] Prior to the start of the patient interviews, the study and all associated patient-related materials were reviewed and approved by the New England Independent Review Board (IRB #120190082). The patient interviews were conducted by phone in April and May 2019. Approximately 1 week prior to the interview, patients were emailed an informed consent form (ICF) and a copy of the ATTR-PSS. Patients were asked to read, sign, and return the ICF in advance of the scheduled interview; it was requested that patients do not review the survey prior to the interview. At the start of the interview, the interviewer provided a brief description of the purpose of the interview, answered any questions on the ICF, and obtained permission from the patient to audio-record the interview.

[378] The interviews followed a semi-structured interview guide. While the interview guide was primarily developed prior to the clinician interviews, it was revised to incorporate questions and probes related to topics that emerged during the clinician interviews. The topics included in the guide are depicted in Figure 4.

[379] The interview started with a brief conversation regarding patients’ experience with ATTR amyloidosis, and then turned to evaluate the ATTR-PSS. This part of the interview utilized cognitive debriefing methodology, which allows participants the opportunity to discuss the relevance of items and their understanding of each aspect of the survey. Patients completed the ATTR-PSS using the think-aloud method [Kucan L, Beck IL. Thinking aloud and reading comprehension research: inquiry, instruction, and social interaction. Rev Educ Res. 1997;67(3):271-299]; through this method, patients verbalize their thoughts while reading each part of the survey and answering all of the survey questions. During this process, patients were asked to describe any aspects of the ATTR-PSS they found challenging or confusing. The interviewer then asked a series of targeted questions about the survey, including its overall relevance and the clarity of instructions, items, recall period, and response choices; patients were asked to comment on each of these and provide feedback on any survey -related topics that had not already been covered.

Data coding and analysis

[380] All interview recordings were transcribed verbatim. Clinician and patient interview data were coded and analyzed separately, but followed the same procedure.

[381] At the conclusion of each interview, a Microsoft Excel spreadsheet was populated with any issues that emerged from the interview that suggested a change be made to the ATTR-PSS. Such issues included survey elements perceived as confusing or difficult to answer, or suggestions provided by interviewees to improve the clarity of the survey. Each unique suggestion was recorded in a single row of the spreadsheet, with a separate column for each interview. As such, the spreadsheet tracked 1) each of the suggestions provided throughout the interviews and 2) the number of interviewees who made the same suggestion. This preliminary coding took place prior to receiving transcripts and was solely based on interviewer notes. Next, transcripts of each interview were reviewed for quality and then cross-checked against the Excel spreadsheet in order to confirm all feedback had been accurately recorded.

[382] After transcripts were reviewed against the information included in the spreadsheet, formal content coding was conducted; all remaining relevant data from the interviews were coded. All coded data were reviewed and analyzed by a primary coder to identify necessary survey modifications. Finally, modifications were made to the survey through a consensus-based approach. Consensus was reached when all team members agreed on the modifications to the survey. Clinician or patient suggestions that were primarily personal preferences for wording or presentation style, or suggestions made by a single individual, generally did not lead to survey modifications. Suggestions aimed at improving the clarity or comprehensiveness of the survey (e.g., modifying symptom descriptions) that were suggested by multiple clinicians or patients were evaluated by the research team and implemented as described in subsequent sections. The research team evaluated the perceived importance of each suggestion and subsequently decided whether a modification was needed. In certain limited instances, the research team was unable to reach consensus regarding whether a modification was necessary, or how a patient’s suggestion should be implemented. When this occurred, the research team reviewed existing literature, meeting minutes from the patient advisory board meeting, and transcripts from the earlier patient focus group to better understand the evolution of the survey items and the totality of evidence in favor of any particular modification. In 1 instance, researchers also contacted the clinicians who participated in interviews to gain additional insight regarding the best way to modify the survey in response to patient suggestions. ¹

[383] The survey was modified twice: once after clinician interviews, and once after patient interviews. As such, patients reviewed a draft of the survey that had been modified as a result of the information obtained from the clinician interviews.

Results

Clinician cognitive debriefing

Clinician demographics

[384] Characteristics of the 7 clinicians who were interviewed are summarized in Table 5. Five hematologists/oncologists, 1 neurologist, and 1 cardio-oncologist were interviewed. All clinicians had at least 5 years of experience treating patients with ATTR amyloidosis, reported experience treating both patients with ATTRm and ATTRwt, and had used PRO measures in clinical practice and/or clinical trials. [385] Table 5. Demographic characteristics of clinicians who participated in cognitive debriefing interviews

ATTR-PSS Cognitive Debriefing: Clinicians (N=7)

N %*

Primary Practice Type

Hematology/Oncology 5 71%

Neurology 1 14%

Cardio-Oncology 1 14%

Gender

Female 3 43%

Male 4 57%

Region of Residence/Practice

Northeast, US 1 14%

Southeast, US 1 14%

Midwest, US 4 57%

Europe 1 14%

Number of years in practice

5-10 years 3 43%

11-20 years 1 14%

21 + years 3 43%

Mean, % Range

Number of patients treated... with ATTR amyloidosis 67 30-100 withATTRm* 25, 38% 7-60 withATTRwt* 36, 61% 15-70

Abbreviations: ATTR-PSS = Amyloid transthyretin amyloidosis patient symptom survey; ATTRm = hereditary ATTR amyloidosis; ATTRwt = Wild type ATTR amyloidosis

*percentages may not sum to 100% due to rounding ^percentage calculated out of all treated patients with ATTR amyloidosis Summary of clinician cognitive debriefing results

[386] Clinicians generally agreed the ATTR-PSS symptom list was relevant, comprehensive, and inclusive of symptoms patients with ATTRm and ATTRwt experience. Clinicians found the order of the list logical and easy to follow. Representative quotations from clinicians regarding their evaluation of different aspects of the ATTR-PSS, and suggested modifications to the survey, are provided in Table 6.

[387] Table 6. Overview of results from clinician cognitive debriefing

Element of ATTR-PSS Representative Clinician Quote

I think it's a very appropriate length. This is not overwhelming. I think it's

Length of survey good. Again, it's all the symptoms that patients experience. I think it's good.

I think it will get really important information that hasn't previously been captured regularly in this patient population. I think we’ll learn new things

Breadth of survey about the extent of their autonomic failure and the symptomatic cardiac content failure that they're having. This is a very good... I think it’ll be important to be included with patients who are on therapy.

In my facilities, we’ve had situations where owing to illiteracy we’ve had caregivers complete the questions and there have been situations where the caregiver would read out the question and the patient would say something,

Instructions but then the caregiver would say, ‘No. I noted when you were short of breath most of the time and not just sometimes. ’...They are the ones completing it for the patient. Again, this might be an anecdote and not really an issue, but it’s something that I’ve experienced.

I think it's good and it's very relevant to the patients. Again, a lot of the research in this field has struggled with how to assess these types of things in these patients. Amyloid is a very different disease from everything else that clinical trials have been done in, and so there's a lot of difficulty in

Symptom list figuring out what we should be assessing. This is a good global assessment of what patients are feeling and I think that's valuable.

Fatigue is a big one. The heart failure symptoms. Anything with shortness of breath, dizziness, edema. The pain ones are very important. Then the nutrition one. The GI symptoms. Yes, it’s gotten them all.

Some of the symptoms, like dementia, I don’t know necessarily the patients can quantify that as rarely, sometimes, most of the time, all the time. Dementia is more of a medical diagnosis the same as sleep apnea. Again, malnutrition is another one that stands out... That they are rarely malnourished or most of the time malnourished. Those seem like more as clinical diagnoses more than symptom of patient and you’re trying to get

Clarity of symptoms patient symptoms. I recommend you being more specific. What it is that you’re trying to get from patients.

I would say, ‘Can you feel hot and cold?’ What I actually ask patients is, ‘You put your foot into a hot bathtub, can you tell you're at risk of burning yourself?’ You have to tell the temperature of the bathtub by your hand because you can't dip your toe and know.

[388] Clinicians confirmed that the ATTR-PSS captured the full extent of the patient experience of ATTR amyloidosis. Initially, 4 clinicians found the survey too long, but after an explanation of planned skip logic designed to alleviate respondent fatigue, 3 of the 4 agreed the length was appropriate. Clinicians were in agreement that the recall period of 1 month is an appropriate timeframe in which to ask patients to reflect back on their symptoms. Two clinicians worried that someone else might complete the form for the patient due to neuropathy in the hands; as a result, an instruction was added stating all answers should reflect the patient experience, and not the impressions or experience of a caregiver. Simple edits (e.g., adding the words ‘each’ and ‘had’) and formatting changes (e.g., underlining) were made to 4 of the items in the ATTR-PSS to improve the clarity of the questions, based on clinicians’ suggestions.

[389] All clinicians identified several conditions on the symptom list they felt were better characterized as medical diagnoses than as symptoms (malnutrition, dementia, sleep apnea, spinal stenosis, carpal tunnel syndrome, stroke, depression, anxiety, and seizures) and as such might be difficult to evaluate on the scales provided (e.g., it would be difficult for a patient to evaluate the frequency of carpal tunnel). Clinicians suggested these be removed from the symptom list, and added to a new item asking if patients had been diagnosed with or experienced any of the conditions. Likewise, clinicians indicated the symptoms of unintentional weight loss and weight gain were difficult to evaluate using the scales provided; these were removed from the symptom list and included as a separate item which asked whether the patient had experienced unintended weight loss or weight gain of 10 or more pounds.

[390] Across clinicians, a total of 12 symptoms were recommended for addition to the ATTR- PSS. Of these 12 symptoms, 4 were recommended for inclusion by multiple clinicians: falls/sudden falling when trying to stand, fecal incontinence, rapid heartbeat/heart palpitations, loss of taste/altered taste. Three of these symptoms were subsequently added to the ATTR-PSS, while the fourth (loss of taste/altered taste) was kept in the survey for interviewers to review with patients.

[391] Clinicians found that, generally, the language used to describe each of the symptoms included in the ATTR-PSS was clear. However, 1 symptom was modified as a result of clinician feedback to more clearly align with the actual patient experience (original description: loss of sensitivity to temperature; revised description: loss of sensitivity to hot and cold). Two other symptoms, sensitivity to alcohol and pain (other than neuropathic pain), were not endorsed by clinicians as part of the ATTR amyloidosis disease experience but were left in the ATTR-PSS and explicitly probed in the patient interviews for clarity and interpretation. Patient cognitive debriefing

Patient demographics

[392] Characteristics of the 10 patients who participated in cognitive debriefing interviews are summarized in Table 7.

[393] Table 7. Demographic characteristics of patients who participated in cognitive debriefing interviews

ATTR-PSS Cognitive Debriefing: Patients (N=10) n %

Gender

Female 5 50%

Male 5 50%

Age range

> 50 0 0%

50-54 2 20%

55-59 4 40%

60-64 1 10%

65-69 2 20%

< 70 1 10%

Type of ATTR

ATTRm with CM 2 20%

ATTRm with PN 4 40%

ATTRm with CM & PN 2 20%

ATTRwt 2 20%

Mean Range

<2 months to

Time since diagnosis 2.38 years >5 years

Abbreviations: ATTR-PSS = Amyloid transthyretin amyloidosis patient symptom survey; CM = cardiomyopathy; ATTRm = hereditary ATTR amyloidosis; ATTRwt =wild type ATTR amyloidosis; PN = peripheral neuropathy

[394] Summary of patient cognitive debriefing results

[395] Overall, patients voiced favorable impressions of the ATTR-PSS and found the length of the survey to be acceptable. Representative quotations from patients regarding their evaluation of the ATTR-PSS are provided in Table 8. [396] Table 8. Overview of results from patient cognitive debriefing

Element of

Representative Patient Quote ATTR-PSS

I didn’t find it too long at all because it’s something that I could walk through fairly quickly. Again, you’re talking about your own symptoms. I know what I

Length of survey feel. I know what’s happening to me. It’s not some kind of nebulous question out there (female, ATTRm)

Because these are actual items that are related to the condition and you want to get Breadth of survey a full understanding of what the patient has and doesn’t have ... I think the survey is content good because had I known that these symptoms attributed to amyloidosis, I would’ve been more aware, or trying to figure it out sooner (female, ATTRm)

I can’t curl the same dumbbell at 80 that I could at 75, but I’m not sure that’s related to amyloidosis as it is to just old age ... Erectile dysfunction or other sexual

Instructions dysfunction... Again, I can’t differentiate between old age and amyloidosis... In other words, I don’t want to attribute it to amyloidosis, when again age is a factor (male, ATTRwt)

Interviewer: Then there is a line that says, ‘Please select up to five symptoms’ which I had indicated that you chose seven. I’m not sure if you saw...

Patient: (Laughs). I didn’t even read that.

Item text Interviewer: That’s what I thought. You missed it.

Patient: Mm-hmm (affirmative). Yes. We need to put that next to the question. Interviewer: Move it up a line?

Patient: Yes, because I went right by that (female, ATTRm)

Pain

‘Pain,’ that is extremely broad and what the heck does that mean, because it's not associated with any one particular thing there, just pain. That's an odd question. In the past month have I experienced pain? I'm irritated by the question. Sorry, I keep thinking, ‘That's entirely too broad. What does pain mean?’ If it was pain that was associated with one particular thing it would be easy. And, also ... I think

Clarity of psychological pain. So, I'm like, ‘What is this?’ (female, ATTRm) symptoms

Sexual dysfunction

You could probably break it up for women and men because, men with erectile would apply and the second part would be for the ladies. I’ve got pain and dryness, so you may want to separate it out for men and women... Because I had to think about it. The first part doesn’t apply to me, the second part, yes, that does (female, ATTRm) Element of Representative Patient Quote ATTR-PSS

Pain, numbness, or tingling in feet or legs

...pain, numbness, or tingling in your feet or legs. I’m going to put most of the time for me. I think that’s a good question. The doctor seems to ask that quite a bit, as well, with the disease, so pretty self-explanatory. Then qualifying it in the feet or legs is good because I think there are other parts, too, that maybe have some (male, ATTRm)

[397] Cognitive debriefing interviews with patients confirmed the relevance of the symptom list and items of the ATTR-PSS, providing positive feedback on the overall comprehensibility of the items, instructions, recall period, and response options.

[398] The greatest difficulty patients experienced while completing the ATTR-PSS was deciding whether or not to endorse a symptom they experience but could not definitively attribute to their ATTR amyloidosis. To address this challenge, relevant text in the instructions for each item were bolded to emphasize that patients should only report on symptoms that are related to ATTR amyloidosis.

[399] Four patients failed to notice that item 3 of the survey asks them to choose only 5 symptoms that have the greatest impact on their daily life. While this could be prevented in an electronic version of the survey through programming logic, additional formatting was also added to the instruction text of this item (e.g., bolded font, revision of line breaks).

[400] Patients recommended edits be made to 6 symptoms of the ATTR-PSS to improve clarity; the wording was revised on 5 of the symptoms, while 1 was removed entirely. Three different pain-related symptoms were originally included in the ATTR-PSS. Two described symptoms of neuropathic pain (‘pain, numbness, or tingling in the feet or legs;’ ‘pain, numbness, or tingling in the arms or hands’), while a third was meant to encompass any other type of painful symptom, and appeared in the symptom list simply as ‘pain.’ While patients understood the 2 symptoms related to neuropathic pains, they (similar to the clinicians) had difficulty understanding how to interpret the third type more ambiguous pain symptom, particularly in comparison to the other specific types of pain on the list. After additional consultation with the clinicians who had participated in the cognitive debriefing interviews, this symptom was ultimately revised to read ‘any other type of pain,’ which was placed after the other two pain- related symptoms and more accurately described pain that was not neuropathic in nature, but could occur in any part of the body. The symptom related to sexual dysfunction was revised with language more clearly inclusive of sexual dysfunction experienced by individuals of any sex rather than having examples that would only be experienced by men. The symptom ‘stress’ was changed to ‘stress due to ATTR amyloidosis,’ to alleviate patient-reported confusion stemming from uncertainty regarding whether they should endorse the symptom if they are experiencing stress due to their condition or due to non-disease-related factors. Minor modifications to the wording of 2 other symptoms were made to increase clarity and facilitate accurate interpretation (‘severe headaches or migraines’ was revised to ‘headaches;’ ‘other (please specify)’ was revised to ‘other symptoms (please specify)’). The symptom ‘sensitivity to alcohol’ was removed entirely from the symptom list, as none of the patients in the study reported experiencing it, and both clinicians and patients expressed confusion regarding how to appropriately interpret its meaning.

[401] In response to patients’ suggestions, two conditions, congestive heart failure and Crohn’s disease, were added to the item asking whether patients had been previously diagnosed with certain medical conditions.

Discussion

[402] This qualitative research study was designed to elicit feedback from both clinicians and patients on the comprehensiveness, comprehensibility, and ease of use of the ATTR-PSS. Due to the varied nature of ATTR amyloidosis, developing a single survey that captures all relevant symptoms while not being overly burdensome for patients to complete is especially challenging. Nevertheless, our study provides strong evidence that the ATTR-PSS overcomes these obstacles. The ATTR-PSS is the first known PRO measure designed specifically for patients with ATTR amyloidosis, with demonstrated evidence of content validity following the FDA’s guidance for measurement development [U.S. Department of Health and Human Services, Food and Drug Administration, supra ; Amyloidosis Research Consortium. Advancing amyloidosis: a research roadmap; 2019. https://www.arci.org/wp-content/uploads/2019/03/ARC-White-Paper-Final- v3.pdf. Accessed August 26, 2019] [403] The cognitive debriefing interviews with both clinicians and patients provided ample evidence that the ATTR-PSS is relevant to patients with different types of ATTR amyloidosis, comprehensive, appropriate, and easy to understand and complete. While the revised survey includes a total of 32 symptoms and 12 other medical diagnoses or complications, the majority of participants found the length of the survey acceptable, especially given the likelihood it will be available as an online survey or mobile application, which will include electronic programming logic to the facilitate survey administration. Importantly, the cognitive debriefing interviews provided evidence to support the use of the ATTR-PSS in patients with ATTRm and ATTRwt, as the symptom list is comprehensive enough to capture symptoms associated with CM, PN, and other organ involvement. Clinicians who reviewed the ATTR-PSS not only agreed that it would be useful to incorporate into clinical trial research, but also acknowledged its potential benefit in a clinical practice setting (especially if it was formatted for electronic administration). Given its potential for widespread use across a variety of contexts, future research should focus on evaluating the psychometric properties of the ATTR-PSS and developing a scoring algorithm. Figure 5 summarizes the modifications made to the ATTR-PSS symptom list as a result of clinician and patient feedback. Conditions listed in ‘medical diagnoses’ and ‘other’ categories were originally included as part the symptom list. At the suggestion of clinicians, these conditions were removed from the symptom list and added as 2 new items to the ATTR-PSS (as represented by the ★ symbol). Revised symptoms (represented by the ★ and § symbols) were modified for language/clarity; the updated language is reflected in the figure. In total, 6 symptom descriptions were revised, 3 symptoms and 2 medical diagnoses were added, and 1 symptom was removed. In addition, weight loss and other complications were removed from the symptom list, and added to 2 new separate items. In total, the 6 revisions and 5 additions accounted for only 25% of all symptoms/conditions included in the revised survey, while the majority of the survey remained unchanged. Together these revisions - along with minor modifications to formatting and instruction text - ensure all relevant symptom-related patient experiences can be reported through the ATTR-PSS, and help increase the ease with which patients can accurately interpret and respond to the survey.

Strengths and limitations [404] This study had several strengths. Among them, the patient sample included both those with ATTRm and ATTRwt. Of the patients with ATTRm, some had symptoms of CM, some had symptoms of PN, and some had symptoms of both, confirming the survey is relevant across patients with mixed phenotype and diverse symptom experiences. Second, the 2-part design of the study allowed for an iterative approach to survey modification. In interviewing clinicians first, researchers were able to modify both the survey and the patient interview guide to take into account feedback from the clinicians. In some cases, feedback from the patients was then presented back to the clinicians before a final determination was made. In this way, it was ensured that modifications to the survey were appropriate and accurately reflected the input of both those living with the disease and those with experience treating the disease.

[405] As with any study, limitations also existed. While collaboration with an advocacy group allowed for relatively fast recruitment of 10 patients who fit both the study’s inclusion criteria and sampling quotas, it is possible this approach also led to the inclusion of more well-informed, engaged participants. All 10 patients were college educated, and 6 of the 10 patients had post graduate degrees, thus their education and literacy levels may be higher than the average patient. As such, aspects of the survey they found understandable or easy to interpret may not be similarly clear to patients with lower literacy levels. In addition, while modifications to the ATTR-PSS based on patient interviews were not major in nature, best practice would suggest the modified ATTR-PSS undergo additional interviews with patients to ensure modifications made did not add complexity or confusion. However, of the 4 symptoms that were revised after the patient interviews, 2 were reviewed by clinicians or subject matter experts to ensure that confusion or unnecessary complexity would not be introduced by the survey modifications.

Conclusion

[406] The ATTR-PSS is an understandable and easy to use assessment of the symptoms of ATTR amyloidosis, and is intended for use in patients regardless of the type of ATTR amyloidosis with which they have been diagnosed. This survey provides a comprehensive evaluation of symptoms and experiences not measured by other PROs. Use of this survey, whether as part of routine clinical care or to measure an endpoint in clinical trials, can help contribute to a more complete assessment of a patient’s health status.

Claims

WHAT IS CLAIMED IS:

1. A method of controlling treatment of subjects having or at risk of a transthyretin amyloidosis comprising monitoring at least one sign and/or symptom of the subjects, wherein the at least one sign or symptom includes at least one of dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition; and initiating or modifying a treatment regimen in at least some of the subjects based on the at least one sign or symptom monitored.

2. The method of claim 1, wherein the monitoring comprises determining the at least one sign or symptom of the subjects before and after initiating or modifying the treatment regimen in at least some of the subjects.

3. The method of claim 1, wherein the treatment regimen comprises administering an antibody specifically binding to TTR.

4. The method of claim 3, wherein the antibody reduces deposits of TTR.

5. The method of any preceding claim, wherein the monitoring monitors at least five of the signs and/or symptoms.

6. The method of claim 5, wherein the monitoring monitors all of the signs and/or symptoms.

7. The method of any preceding claim, wherein the monitoring also monitors one or more signs and/or symptoms selected from fatigue; shortness of breath; dizziness or fainting; chest pain; sleep disturbance; pain, numbness or tingling in feet or legs; pain, numbness or tingling in hands or arms; loss of sensitivity to temperature; swelling of legs or ankles; muscle weakness or loss of strength; pain; carpal tunnel syndrome; feeling full quickly when eating; nausea; vomiting; weight loss; diarrhea; constipation; urinary incontinence; blurred vision; sexual dysfunction including erectile dysfunction, vaginal pain or dryness, decreased libido, or other sexual dysfunction; blood in urine; spinal stenosis; stress; anxiety; depression and sensitivity to alcohol.

8. The method of claim 7, wherein the monitoring also monitors at least 5, 10, 15, 20, 25 or all of the signs and/or symptoms.

9. The method of any preceding claim further comprising calculating an index of the subjects’ conditions from the monitored signs and/or symptoms.

10. The method of any preceding claim, wherein the monitoring further determines a subset of the monitored signs and/or symptoms having most impact on daily life of the subjects.

11. The method of any preceding claim, wherein the monitoring further determines severity of the monitored signs and/or symptoms.

12. The method of any preceding claim, wherein the monitoring further determines frequency of the monitored signs and/or symptoms.

13. The method of claim 9, wherein the initiating or modifying of the treatment regimen is based on the value of the index in a subject relative to one or more reference values.

14. The method of any preceding claim, wherein the monitoring is performed weekly, monthly, quarterly, every six months or every year.

15. The method of claim 9, wherein the treatment regimen is initiated when the index equals or exceeds a reference value from subjects with TTR amyloidosis.

16. The method of claim 9, wherein the treatment regimen is initiated when the index differs by at least two standard deviations from the value in control subjects.

17. The method of any preceding claim, wherein the subjects have TTR- amyloidosis and receive a treatment regimen comprising an antibody against TTR and the treatment regimen is modified based on the monitoring.

18. The method of claim 17, wherein the dose or frequency of administration of the antibody is modified based on the monitoring.

19. The method of any one of claims 1-16, wherein the subjects have or are at risk of TTR amyloidosis, and a treatment regimen comprising an antibody against TTR is initiated based on the monitoring.

20. The method of any one of claims 1-16, wherein the subjects have TTR amyloidosis and are receiving a treatment regimen other than an antibody against TTR, wherein the treatment regimen is discontinued and replaced with a treatment regimen comprising an antibody against TTR based on the monitoring.

21. The method of any preceding claim, wherein the subjects are diagnosed with TTR amyloidosis by presence of TTR deposits or a TTR level in blood.

22. The method of claim 21, wherein diagnosis of TTR amyloidosis is also based on a genetic mutation in a gene encoding TTR.

23. A method of treating a subject having or at risk of a transthyretin amyloidosis comprising administering an antibody specifically binding to TTR to the subject, wherein signs and/or symptoms of the subject are monitored and the signs and/or symptoms include at least one of dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition; and initiating or modifying a treatment regimen in the subject based on the symptoms.

24. A method of treating a subject having or at risk of a transthyretin amyloidosis comprising administering an antibody specifically binding to TTR to the subject, and monitoring a response of the subject to the administration from signs and/or symptoms of the subject, wherein the signs and/or symptoms include at least one of dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition.

25. A method of monitoring a subject having or at risk of a transthyretin amyloidosis and receiving an antibody specifically binding to TTR for treatment or prophylaxis of the amyloidosis, the method comprising monitoring signs and/or symptoms of the subject, wherein the signs and/or symptoms include at least one of dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition.

26. A method of controlling treatment of a subject having or at risk of a transthyretin amyloidosis comprising monitoring signs and/or symptoms of the subject, wherein the signs and/or symptoms include at least one of dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition; and initiating or modifying a treatment regimen of the subject based on the symptoms.

27. The method of claim 26, wherein the monitoring comprises determining signs and/or symptoms of the subject before and after initiating or modifying the treatment regimen.

28. The method of claim 26, wherein the treatment regimen comprises administering an antibody specifically binding to TTR.

29. The method of claim 28, wherein the antibody reduces deposits of TTR.

30. The method of any one of claims 26-29, wherein the monitoring monitors at least five of the signs and/or symptoms.

31. The method of claim 30, wherein the monitoring monitors all of the signs and/or symptoms.

32. The method of any preceding claim, wherein the monitoring also monitors one or more signs and/or symptoms selected from fatigue; shortness of breath; dizziness or fainting; chest pain; sleep disturbance; pain, numbness or tingling in feet or legs; pain, numbness or tingling in hands or arms; loss of sensitivity to temperature; swelling of legs or ankles; muscle weakness or loss of strength; pain; carpal tunnel syndrome; feeling full quickly when eating; nausea; vomiting; weight loss; diarrhea; constipation; urinary incontinence; blurred vision; sexual dysfunction including erectile dysfunction, vaginal pain or dryness, decreased libido or other sexual dysfunction; blood in urine; spinal stenosis; stress; anxiety; depression and sensitivity to alcohol.

33. The method of claim 32 wherein the monitoring also monitors at least 5, 10, 15, 20, 25 or all of the signs and/or symptoms.

34. The method of any one of claims 26-33 further comprising calculating an index of the subject’s conditions from the monitored signs and/or symptoms.

35. The method of any one of claims 26-34, wherein the monitoring further determines a subset of the monitored signs and/or symptoms having most impact on daily life of the subject.

36. The method of any one of claims 26-35, wherein the monitoring further determines severity of the monitored signs and/or symptoms.

37. The method of any one of claims 26-36, wherein the monitoring further determines frequency of the monitored signs and/or symptoms.

38. The method of claim 34, wherein the initiating or modifying of the treatment regimen is based on the value of the index in a subject relative to one or more reference values.

39. The method of any one of claims 26-33, wherein the monitoring is performed weekly, monthly, quarterly, every six months or every year.

40. The method of claim 34, wherein the treatment regimen is initiated when the index equals or exceeds a reference value from subjects with TTR amyloidosis.

41. The method of claim 34, wherein the treatment regimen is initiated when the index differs by at least two standard deviations from the value in control subjects.

42. The method of any one of claims 26-34, wherein the subject has TTR- amyloidosis and receives a treatment regimen comprising an antibody against TTR and the treatment regimen is modified based on the monitoring.

43. The method of claim 42, wherein the dose or frequency of administration of the antibody is modified based on the monitoring.

44. The method of any one of claims 26-41, wherein the subject has or is at risk of TTR amyloidosis, and a treatment regimen comprising an antibody against TTR is initiated based on the monitoring.

45. The method of any one of claims 26-45, wherein the subject has TTR amyloidosis and is receiving a treatment regimen other than an antibody against TTR, wherein the treatment regimen is discontinued and replaced with a treatment regimen comprising an antibody against TTR based on the monitoring.

46. The method of any one of claims 26-45, wherein the subject is diagnosed with TTR amyloidosis by presence of TTR deposits or a TTR level in blood.

47. The method of claim 46, wherein diagnosis of TTR amyloidosis is also based on a genetic mutation in a gene encoding TTR.

48. A method of diagnosing TTR amyloidosis in a subject, the method comprising:

(a) monitoring signs and/or symptoms of the subject, wherein the signs and/or symptoms include at least one of dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues, weight gain, sleep apnea, cognitive issues, rapid heartbeat or heart palpitations, falling or a sudden fall when trying to stand, fecal incontinence, congestive heart failure, Crohn’s disease, and malnutrition; and

(b) diagnosing TTR amyloidosis in the subject based on the at least one sign or symptom monitored.

49. The method of claim 48, wherein the monitoring also monitors one or more signs and/or symptoms selected from fatigue; shortness of breath; dizziness or fainting; chest pain; sleep disturbance; pain, numbness or tingling in feet or legs; pain, numbness or tingling in hands or arms; loss of sensitivity to temperature; swelling of legs or ankles; muscle weakness or loss of strength; pain; carpal tunnel syndrome; feeling full quickly when eating; nausea; vomiting; weight loss; diarrhea; constipation; urinary incontinence; blurred vision; sexual dysfunction including erectile dysfunction, vaginal pain or dryness, decreased libido or other sexual dysfunction; blood in urine; spinal stenosis; stress; anxiety; depression and sensitivity to alcohol.

50. The method of claim 48 or claim 49, wherein the diagnosing of TTR amyloidosis is also based on presence of TTR deposits or a TTR level in blood.

51. The method of claim 48 or claim 49, wherein the diagnosing of TTR amyloidosis is also based on presence of a genetic mutation in a gene encoding TTR.

52. The method of claim 48 or claim 49, wherein the diagnosing of TTR amyloidosis is also based on imaging by electrocardiogram or MRI.