WO2021168156A1 - Surveillance de l'amyloïdose de la transthyrétine - Google Patents

Surveillance de l'amyloïdose de la transthyrétine Download PDF

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Publication number
WO2021168156A1
WO2021168156A1 PCT/US2021/018632 US2021018632W WO2021168156A1 WO 2021168156 A1 WO2021168156 A1 WO 2021168156A1 US 2021018632 W US2021018632 W US 2021018632W WO 2021168156 A1 WO2021168156 A1 WO 2021168156A1
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WIPO (PCT)
Prior art keywords
ttr
symptoms
antibody
amyloidosis
monitoring
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PCT/US2021/018632
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English (en)
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Tiffany P. QUOCK
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Prothena Biosciences Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/40—Detecting, measuring or recording for evaluating the nervous system
    • A61B5/4076—Diagnosing or monitoring particular conditions of the nervous system
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H20/00—ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance
    • G16H20/10—ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance relating to drugs or medications, e.g. for ensuring correct administration to patients
    • G16H20/17—ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance relating to drugs or medications, e.g. for ensuring correct administration to patients delivered via infusion or injection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/30—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for calculating health indices; for individual health risk assessment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B6/00—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment
    • A61B6/02—Arrangements for diagnosis sequentially in different planes; Stereoscopic radiation diagnosis
    • A61B6/03—Computed tomography [CT]
    • A61B6/037—Emission tomography
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39583—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials not provided for elsewhere, e.g. haptens, coenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7047—Fibrils-Filaments-Plaque formation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • Transthyretin is one of the many proteins that are known to misfold and aggregate (e.g., undergo amyloidogenesis).
  • Transthyretin-mediated amyloidosis 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.
  • familial disease arising from misfolding of a mutated or variant TTR
  • 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.
  • 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.
  • 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
  • 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.
  • the treatment regimen comprises administering an antibody specifically binding to TTR.
  • the antibody reduces deposits of TTR.
  • the monitoring monitors at least five of the signs and/or symptoms.
  • the monitoring monitors all of the signs and/or symptoms.
  • 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.
  • the monitoring also monitors at least 5, 10, 15, 20, 25 or all of the signs and/or symptoms.
  • the method further comprises calculating an index of the subjects’ conditions from the monitored signs and/or symptoms.
  • the monitoring further determines a subset of the monitored signs and/or symptoms having most impact on daily life of the subjects.
  • the monitoring further determines severity of the monitored signs and/or symptoms.
  • the monitoring further determines frequency of the monitored signs and/or symptoms.
  • 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.
  • the monitoring is performed weekly, monthly, quarterly, every six months or every year.
  • the treatment regimen is initiated when the index equals or exceeds a reference value from subjects with TTR amyloidosis.
  • the treatment regimen is initiated when the index differs by at least two standard deviations from the value in control subjects.
  • 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.
  • the dose or frequency of administration of the antibody is modified based on the monitoring.
  • 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.
  • 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.
  • the subjects are diagnosed with TTR amyloidosis by presence of TTR deposits or a TTR level in blood.
  • diagnosis of TTR amyloidosis is also based on a genetic mutation in a gene encoding TTR.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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 incontine
  • the monitoring comprises determining signs and/or symptoms of the subject before and after initiating or modifying the treatment regimen.
  • the treatment regimen comprises administering an antibody specifically binding to TTR.
  • the antibody reduces deposits of TTR.
  • the monitoring monitors at least five of the signs and/or symptoms.
  • the monitoring monitors all of the signs and/or symptoms.
  • 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.
  • 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
  • the monitoring also monitors at least 5, 10, 15, 20, 25 or all of the signs and/or symptoms.
  • the method further comprises calculating an index of the subject’s conditions from the monitored signs and/or symptoms. [13]
  • the monitoring further determines a subset of the monitored signs and/or symptoms having most impact on daily life of the subject.
  • the monitoring further determines severity of the monitored signs and/or symptoms.
  • the monitoring further determines frequency of the monitored signs and/or symptoms.
  • 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.
  • the monitoring is performed weekly, monthly, quarterly, every six months or every year.
  • the treatment regimen is initiated when the index equals or exceeds a reference value from subjects with TTR amyloidosis.
  • the treatment regimen is initiated when the index differs by at least two standard deviations from the value in control subjects.
  • 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.
  • the dose or frequency of administration of the antibody is modified based on the monitoring.
  • 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.
  • a treatment regimen comprising an antibody against TTR is initiated based on the monitoring.
  • 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.
  • the subject is diagnosed with TTR amyloidosis by presence of TTR deposits or a TTR level in blood.
  • diagnosis of TTR amyloidosis is also based on a genetic mutation in a gene encoding TTR.
  • 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.
  • 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
  • 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.
  • 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
  • the diagnosing of TTR amyloidosis is also based on presence of TTR deposits or a TTR level in blood.
  • the diagnosing of TTR amyloidosis is also based on presence of a genetic mutation in a gene encoding TTR.
  • the diagnosing of TTR amyloidosis is also based on imaging by electrocardiogram or MRI.
  • SEQ ID NO: 1 sets forth the amino acid sequence of a heavy chain variable region of the mouse 18C5 antibody with signal peptide.
  • SEQ ID NO:2 sets forth a nucleic acid sequence encoding a heavy chain variable region of the mouse 18C5 antibody with signal peptide.
  • SEQ ID NO:3 sets forth the amino acid sequence of a light chain variable region of the mouse 18C5 antibody with signal peptide.
  • SEQ ID NO:4 sets forth a nucleic acid sequence encoding a light chain variable region of the mouse 18C5 antibody with signal peptide.
  • SEQ ID NO:5 sets forth the amino acid sequence of a Kabat/Chothia Composite CDR-H1 of the mouse 18C5 antibody.
  • SEQ ID NO:6 sets forth a nucleic acid sequence encoding a Kabat/Chothia Composite CDR-H1 of the mouse 18C5 antibody.
  • SEQ ID NO:7 sets forth the amino acid sequence of a Kabat/Chothia Composite CDR-H2 of the mouse 18C5 antibody.
  • SEQ ID NO:8 sets forth a nucleic acid sequence encoding a Kabat/Chothia Composite CDR-H2 of the mouse 18C5 antibody.
  • SEQ ID NO:9 sets forth the amino acid sequence of a Kabat/Chothia Composite CDR-H3 of the mouse 18C5 antibody.
  • SEQ ID NO: 10 sets forth a nucleic acid sequence encoding a Kabat/Chothia Composite CDR-H3 of the mouse 18C5 antibody.
  • SEQ ID NO: 11 sets forth the amino acid sequence of a Kabat/Chothia Composite CDR- L1 of the mouse 18C5 antibody.
  • SEQ ID NO: 12 sets forth a nucleic acid sequence encoding a Kabat/Chothia Composite CDR-L1 of the mouse 18C5 antibody.
  • SEQ ID NO: 13 sets forth the amino acid sequence of a Kabat/Chothia Composite CDR- L2 of the mouse 18C5 antibody.
  • SEQ ID NO: 14 sets forth a nucleic acid sequence encoding a Kabat/Chothia Composite CDR-L2 of the mouse 18C5 antibody.
  • SEQ ID NO: 15 sets forth the amino acid sequence of a Kabat/Chothia Composite CDR- L3 of the mouse 18C5 antibody.
  • SEQ ID NO: 16 sets forth a nucleic acid sequence encoding the a Kabat/Chothia Composite CDR-L3 of the mouse 18C5 antibody.
  • SEQ ID NO: 17 sets forth the amino acid sequence of a chimeric 18C5 heavy chain constant region (human IgGl).
  • 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).
  • SEQ ID NO: 19 sets forth the amino acid sequence of a chimeric 18C5 light chain constant region (human kappa).
  • 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).
  • SEQ ID NO:21 sets forth the amino acid sequence of an exemplary IgGl heavy chain constant region.
  • SEQ ID NO:22 sets forth the amino acid sequence of an exemplary IgGl Glm3 heavy chain constant region.
  • SEQ ID NO:23 sets forth the amino acid sequence of an exemplary IgGl Glm3 heavy chain constant region.
  • SEQ ID NO:24 sets forth the amino acid sequence of an exemplary light chain constant region with N-terminal Arginine.
  • SEQ ID NO:25 sets forth the amino acid sequence of an exemplary light chain constant region without N-terminal Arginine.
  • SEQ ID NO:26 sets forth the amino acid sequence of human transthyretin set forth in accession number P02766.1 (UniProt).
  • SEQ ID NO:27 sets forth the amino acid sequence of human transthyretin set forth in accession number AAB35639.1 (GenBank).
  • SEQ ID NO:28 sets forth the amino acid sequence of human transthyretin set forth in accession number AAB35640.1 (GenBank).
  • SEQ ID NO:29 sets forth the amino acid sequence of human transthyretin set forth in accession number and AB 163351.1 (GenBank).
  • SEQ ID NO:30 sets forth the amino acid sequence of residues 101-109 of human transthyretin.
  • SEQ ID NO:31 sets forth the amino acid sequence of residues 87-127 of human transthyretin.
  • SEQ ID NO:32 sets forth a nucleic acid sequence encoding an exemplary IgGl Glm3 heavy chain constant region.
  • SEQ ID NO:33 sets forth a nucleic acid sequence encoding an exemplary light chain constant region with C-terminal Arginine.
  • SEQ ID NO:34 sets forth a nucleic acid sequence encoding an exemplary light chain constant region without C-terminal Arginine.
  • SEQ ID NO:35 sets forth the amino acid sequence of a heavy chain constant region signal peptide.
  • SEQ ID NO:36 sets forth a nucleic acid sequence encoding a heavy chain constant region signal peptide.
  • SEQ ID NO:37 sets forth the amino acid sequence of a light chain constant region signal peptide.
  • SEQ ID NO:38 sets forth a nucleic acid sequence encoding a light chain constant region signal peptide.
  • SEQ ID NO:39 sets forth the amino acid sequence of a Kabat CDR-H1 of antibody 14G8
  • SEQ ID NO:40 sets forth the amino acid sequence of a Kabat CDR-H2 of antibody 14G8
  • SEQ ID NO:41 sets forth the amino acid sequence of a Kabat CDR-H3 of antibody 14G8
  • SEQ ID NO:42 sets forth the amino acid sequence of a Kabat CDR-L1 of antibody 14G8.
  • SEQ ID NO:43 sets forth the amino acid sequence of a Kabat CDR-L2 of antibody 14G8.
  • SEQ ID NO:44 sets forth the amino acid sequence of a Kabat CDR-L3 of antibody 14G8.
  • SEQ ID NO:45 sets forth the amino acid sequence of an epitope of antibody 5A1.
  • SEQ ID NO:46 sets forth the amino acid sequence of a Kabat CDR-H1 of antibody 5A1.
  • SEQ ID NO:47 sets forth the amino acid sequence of a Kabat CDR-H2 of antibody 5A1.
  • SEQ ID NO:48 sets forth the amino acid sequence of a Kabat CDR-H3 of antibody 5A1.
  • SEQ ID NO:49 sets forth the amino acid sequence of a Kabat CDR-L1 of antibody 5A1.
  • SEQ ID NO:50 sets forth the amino acid sequence of a Kabat CDR-L2 of antibody 5A1.
  • SEQ ID NO:51 sets forth the amino acid sequence of a Kabat CDR-L3 of antibody 5A1.
  • SEQ ID NO:52 sets forth the amino acid sequence of a Kabat CDR-H1 of antibody 6C1.
  • SEQ ID NO:53 sets forth the amino acid sequence of a Kabat CDR-H2 of antibody 6C1.
  • SEQ ID NO:54 sets forth the amino acid sequence of a Kabat CDR-H3 of antibody 6C1.
  • SEQ ID NO: 55 sets forth the amino acid sequence of a Kabat CDR-L1 of antibody 6C1.
  • SEQ ID NO:56 sets forth the amino acid sequence of a Kabat CDR-L2 of antibody 6C1.
  • SEQ ID NO:57 sets forth the amino acid sequence of a Kabat CDR-L3 of antibody 6C1.
  • SEQ ID NO:58 sets forth the amino acid sequence of a VH region of antibody AD7F6.
  • SEQ ID NO:59 sets forth the amino acid sequence of a VL region of antibody AD7F6.
  • SEQ ID NO:60 sets forth the amino acid sequence of a CDR-H1 of antibody RT24.
  • SEQ ID NO:61 sets forth the amino acid sequence of a CDR-H2 of antibody RT24.
  • SEQ ID NO:62 sets forth the amino acid sequence of a CDR-H3 of antibody RT24.
  • EQ ID NO:63 sets forth the amino acid sequence of a CDR-L1 of antibody RT24.
  • SEQ ID NO:64 sets forth the amino acid sequence of a CDR-L2 of antibody RT24.
  • SEQ ID NO:65 sets forth the amino acid sequence of a CDR-L3 of antibody RT24.
  • SEQ ID NO:66 sets forth the amino acid sequence of a CDR-H1 of antibody NI-
  • SEQ ID NO: 67 sets forth the amino acid sequence of a CDR-H2 of antibody NI- 301.35G11.
  • SEQ ID NO: 68 sets forth the amino acid sequence of a CDR-H3 of antibody NI- 301.35G11.
  • SEQ ID NO:69 sets forth the amino acid sequence of a CDR-L1 of antibody NI- 301.35G11.
  • SEQ ID NO:70 sets forth the amino acid sequence of a CDR-L2 of antibody NI- 301.35G11.
  • SEQ ID NO:71 sets forth the amino acid sequence of a CDR-L3 of antibody NI- 301.35G11.
  • SEQ ID NO: 72 sets forth the amino acid sequence of an epitope of antibodies MFD101, MDF102, MFD103, MFD105.
  • SEQ ID NO: 73 sets forth the amino acid sequence of an epitope of antibodies MFD 107, MFD108, MFD 109, MFD111.
  • SEQ ID NO:74 sets forth the amino acid sequence of an epitope of antibody MFD114.
  • SEQ ID NO:75 sets forth the amino acid sequence of a Kabat CDR-H1 of antibody 9D5.
  • SEQ ID NO:76 sets forth the amino acid sequence of a Kabat CDR-H2 of antibody 9D5.
  • SEQ ID NO:77 sets forth the amino acid sequence of a Kabat CDR-H3 of antibody 9D5.
  • SEQ ID NO:78 sets forth the amino acid sequence of a Kabat CDR-L1 of antibody 9D5.
  • SEQ ID NO:79 sets forth the amino acid sequence of a Kabat CDR-L2 of antibody 9D5.
  • SEQ ID NO:80 sets forth the amino acid sequence of a Kabat CDR-L3 of antibody 9D5.
  • SEQ ID NO:81 sets forth the amino acid sequence of a mature heavy chain variable region of the mouse 18C5 antibody.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO:85 sets forth the amino acid sequence of a heavy chain variable region of the humanized 18C5 antibody hul8C5-VH_l.
  • SEQ ID NO:86 sets forth the amino acid sequence of a heavy chain variable region of the humanized 18C5 antibody hul8C5-VH_2.
  • SEQ ID NO:87 sets forth the amino acid sequence of a mature light chain variable region of the mouse 18C5 antibody.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO:91 sets forth the amino acid sequence of a light chain variable region of the humanized 18C5 antibody hul8C5-VL_l.
  • SEQ ID NO:92 sets forth the amino acid sequence of a light chain variable region of the humanized 18C5 antibody hul8C5-VL_2.
  • SEQ ID NO:93 sets forth the amino acid sequence of Kabat CDR-H1 of the mouse 18C5 antibody.
  • SEQ ID NO:94 sets forth the amino acid sequence of Chothia CDR-H1 of the mouse 18C5 antibody.
  • SEQ ID NO:95 sets forth the amino acid sequence of Contact CDR-H1 of the mouse 18C5 antibody.
  • SEQ ID NO:96 sets forth the amino acid sequence of Chothia CDR-H2 of the mouse 18C5 antibody.
  • SEQ ID NO:97 sets forth the amino acid sequence of AbM CDR-H2 of the mouse 18C5 antibody.
  • SEQ ID NO: 98 sets forth the amino acid sequence of Contact CDR-H2 of the mouse 18C5 antibody.
  • SEQ ID NO:99 sets forth the amino acid sequence of Contact CDR-H3 of the mouse 18C5 antibody.
  • SEQ ID NO: 100 sets forth the amino acid sequence of Contact CDR-L1 of the mouse 18C5 antibody.
  • SEQ ID NO: 101 sets forth the amino acid sequence of Contact CDR-L2 of the mouse 18C5 antibody.
  • SEQ ID NO: 102 sets forth the amino acid sequence of Contact CDR-L3 of the mouse 18C5 antibody.
  • SEQ ID NO: 103 sets forth the amino acid sequence of a heavy chain variable region of the mouse 9D5 antibody.
  • SEQ ID NO: 104 sets forth the amino acid sequence of a light chain variable region of the mouse 9D5 antibody.
  • 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.
  • Specific binding of an antibody to its target antigen means an affinity of at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • an antibody 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • IGF insulin-like growth factor
  • leptin receptor a leptin receptor
  • lipoprotein receptor or a transferrin receptor
  • 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).
  • 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-IgTM) 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
  • bispecific antibodies examples include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgGl (Xencor) or DuoBody (based on Fab arm exchange, Genmab).
  • 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.
  • 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.
  • TTR transthyretin
  • 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).
  • 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.
  • 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.
  • TTR transthyretin
  • 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,
  • SEQ ID NO:26 103-104, 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.
  • 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.
  • 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.
  • 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 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.
  • TTR transthyretin
  • TTR transthyretin
  • 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.
  • TTR misfolded 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.
  • 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.
  • 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.
  • subject and “patient” are used interchangeably to include human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
  • 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.
  • risk-factor e.g., genetic, biochemical, family history, and situational exposure
  • 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.
  • 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.
  • 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.
  • TTR transthyretin
  • Control samples can be obtained from individuals not afflicted with a TTR amyloidosis or a specifically chosen type of TTR amyloidosis.
  • 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).
  • 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.
  • the tissue in the control sample is the same type as the tissue in the biological sample (e.g., cardiomyocytes in the heart).
  • 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.
  • 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.
  • 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.
  • 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.
  • a subject antibody region e.g ., the entire mature variable region of a heavy or light chain
  • compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited.
  • a composition that “comprises” or “includes” an antibody may contain the antibody alone or in combination with other ingredients.
  • Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.
  • 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.
  • a treatment is symptomatic if it only affects one or more symptoms of a disease, not its cause, i.e., its etiology.
  • 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.
  • Transthyretin 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.
  • TTR thyroxine
  • T4 thyroxine
  • amyloid diseases The established function of TTR in the blood is to transport holo-retinol binding protein.
  • 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.
  • 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.
  • transthyretin or its fragments or domains includes the natural human amino acid sequences including isoforms, mutants (e.g., E89K and E89Q), and allelic variants thereof.
  • 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.
  • 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).
  • TTR amyloidoses encompass diseases caused by pathogenic misfolded TTR resulting from mutations in TTR or resulting from non- mutated, misfolded TTR.
  • wild-type ATTR amyloidosis also called senile systemic amyloidosis or SSA
  • SCA senile cardiac amyloidosis
  • SSA senile systemic amyloidosis
  • SCA senile cardiac amyloidosis
  • TTR amyloidosis is also the most common form of hereditary (familial) amyloidosis, which is caused by mutations that destabilize the TTR protein.
  • 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).
  • FAP familial amyloid polyneuropathy
  • FAC familial amyloid cardiomyopathy
  • CNSA rare central nervous system selective amyloidosis
  • 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).
  • TTR amyloidogenesis 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • AL amyloid light-chain
  • TTR 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
  • the therapy or treatment for treating TTR amyloidosis is selected from the group comprising small molecules, antisense oligonucleotides, small interfering RNAs, and antibodies.
  • the TTR amyloidosis therapy stabilizes TTR, including TTR tetramers.
  • the TTR amyloidosis therapy inhibits TTR protein synthesis.
  • the TTR amyloidosis therapy degrades mutant and wild-type TTR mRNA through RNA interference.
  • the TTR amyloidosis therapy reduces serum TTR protein and TTR deposits in tissue.
  • 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.
  • the TTR amyloidosis treatment is VYNDAQEL® (tafamidis meglumine), VYNDAMAXTM (tafamidis), ONPATTROTM (patisiran), TEGSEDITM (inotersen), diflunisal, doxycycline, tauroursodeoxycholic, tolcapone, an anti-serum amyloid P agent, or an anti-TTR antibody, including those described herein.
  • TTR transthyretin
  • 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.
  • TTR transthyretin
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Antibodies having the binding specificity of a selected murine antibody 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
  • 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 10 8 and preferably at least 10 9 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.
  • 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.
  • 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.
  • 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.
  • Kabat CDRs (CDR-H1, CDR-H2, CDR-H3) of the heavy chain of 9D5 are designated SEQ ID NOs: 75, 76, and 77, respectively
  • Kabat CDRs (CDR-L1, CDR-L2, CDR-L3) of the light chain of 9D5 are designated SEQ ID NOs: 78, 79, and 80, respectively.
  • 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
  • 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.
  • 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.
  • non-human antibodies e.g., murine, guinea pig, primate, rabbit or rat
  • monomeric TTR or a fragment thereof e.g, amino acid residues 89-97 or amino acid residues 101-109
  • Such an immunogen can be obtained from a natural source, by peptide synthesis, or by recombinant expression.
  • the immunogen can be administered fused or otherwise complexed with a carrier protein.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an antibody 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.
  • 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.
  • 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).
  • CDRs 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.,
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the human framework amino acid 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:
  • CDR region e.g ., is within about 6 A of a CDR region
  • a CDR region e.g., identified by modeling the light or heavy chain on the solved structure of a homologous known immunoglobulin chain
  • 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)).
  • 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)].
  • 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.
  • Exemplary humanized antibodies are humanized forms of the mouse 18C5, designated Hul8C5.
  • 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-
  • the invention further provides 2 exemplified human mature light chain variable regions: hul8C5-VL_vl and hul8C5-VL_v2.
  • 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).
  • 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.
  • the first mentioned residue is human
  • the first mentioned residue is mouse.
  • 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.
  • 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.
  • 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
  • H94 is occupied by S or R.
  • 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.
  • 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.
  • positions L2 and L45 in the VL region are occupied by V and R, respectively, as in hul8C5-VL_v2.
  • variable heavy chain has > 85% identity to human sequence.
  • variable light chain has > 85% identity to human sequence.
  • each of the variable heavy chain and variable light chain has > 85% identity to human germline sequence.
  • 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.
  • 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.
  • variable region frameworks 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.
  • Hu9D5 or Hul8C5variants 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.
  • Some methods of the invention use chimeric and veneered forms of non-human antibodies, particularly the 9D5 or 18C5 antibodies of the examples.
  • 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.
  • 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,
  • 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.
  • 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.
  • a fragment of TTR such as a TTR variant containing only amino acid residues 89-97 or amino acid residues 101-109 of TTR
  • 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.
  • 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.
  • 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.
  • One or several amino acids at the amino or carboxy terminus of the light and/or 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.
  • ADCC complement-mediated cytotoxicity
  • 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
  • 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.
  • positions 234, 236 and/or 237 in human IgG2 are substituted with alanine and position 235 with glutamine (see, e.g, US
  • 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.
  • a mutation at one or more of positions 318, 320, and 322 by EU numbering of human IgGl is used.
  • positions 234 and/or 235 are substituted with alanine and/or position 329 is substituted with glycine.
  • positions 234 and 235 are substituted with alanine, such as in SEQ ID NO:23.
  • the isotype is human IgG2 or IgG4.
  • 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.
  • 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.
  • 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.
  • a number of methods are known for producing chimeric and humanized antibodies using an antibody-expressing cell line (e.g hybridoma).
  • the immunoglobulin variable regions of antibodies can be cloned and sequenced using well known methods.
  • 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”).
  • 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.
  • the light chain variable VL region can be cloned in an analogous manner.
  • 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.
  • 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.
  • 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.
  • a mammalian expression vector such as pCMV- hyl for the heavy chain and pCMV-Mcl for the light chain.
  • These vectors encode human g ⁇ and Ck constant regions as exonic fragments downstream of the inserted variable region cassette.
  • 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.
  • 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.
  • 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.
  • selection markers e.g., ampicillin resistance or hygromycin resistance
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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)).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • either the antibody or TTR target is immobilized in such assay.
  • 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.
  • TTR monomeric, misfolded, aggregated, or fibril forms of TTR
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • IONIS TTRRx inotersen
  • siRNAs such as patisiran or revusiran
  • doxycycline doxy
  • TUDCA tauroursodeoxycholic acid
  • CyD cyclodextrin
  • IDOX 4'-iodo-4'-deoxydoxorubicin
  • EGCG epigallocatechin
  • TTR amyloidosis or signs and/or symptoms of a TTR amyloidosis.
  • 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.
  • Antibodies can also be coupled with other proteins.
  • 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).
  • 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.
  • 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.
  • 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 yttrium 90 (90Y), radiosilver-111, radiosilver- 199, Bismuth 213 , iodine ( 131 I, 125 I, 123 I, 121 I,), carbon ( 14 C), sulfur
  • 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).
  • electrochemiluminescent labels for example MSD GOLD SULFO- TAG NHS-Ester (SULFO-TAG) (Meso Scale Diagnostics, Rockville, MD).
  • Linkage of radioisotopes to antibodies may be performed with conventional bifunction chelates.
  • sulfur-based linkers may be used for radiosilver- 111 and radiosilver- 199 linkage. See Hazra et ah, Cell Biophys. 24-25:1-7 (1994).
  • Linkage of silver radioisotopes may involve reducing the immunoglobulin with ascorbic acid.
  • ibritumomab tiuxetan 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).
  • 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.
  • an intermediate e.g a linker
  • 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.
  • 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.
  • TTR transthyretin
  • 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.
  • 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.
  • 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.
  • an exemplary dosing frequency is daily to monthly, although more or less frequent dosing is also possible.
  • 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.
  • TTR transthyretin
  • diseases or conditions mediated at least in part by transthyretin 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).
  • FAC familial amyloid cardiomyopathy
  • FAP familial amyloid polyneuropathy
  • CNSA central nervous system selective amyloidosis
  • 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.
  • 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.
  • 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.,
  • 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.
  • TTR precursor protein responsible for amyloid formation is indeed TTR.
  • immunohistochemical staining and mass spectroscopic identification of TTR are subsequently performed to ensure that the precursor protein responsible for amyloid formation is indeed TTR.
  • demonstration of a mutation in the gene encoding TTR is then needed before a definitive diagnosis can be made.
  • 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.
  • 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.
  • 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.
  • a therapeutic agent e.g., a truncated form of TTR comprising amino acid residues 89-97 or comprising amino acid residues 101-109
  • 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.
  • a disease e.g., TTR amyloidosis
  • 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.
  • 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.
  • a controlled clinical trial e.g, a phase II, phase II/III, or phase III trial
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • 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.
  • agents can include siRNA to inhibit expression of TTR or Vyndaqel, a stabilizer of TTR in tetramer formation.
  • 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.
  • 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).
  • doxy doxycycline
  • TUDCA tauroursodeoxycholic acid
  • CyD cyclodextrin
  • IDOX 4'-iodo-4'-deoxydoxorubicin
  • SAP serum amyloid P component
  • 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.
  • 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.
  • 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.
  • TTR amyloid levels can be measured by a number of methods, including imaging techniques.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • Changes in a subject’s signs and/or symptoms can be used in determining when to initiate treatment of a subject.
  • 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.
  • 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.
  • monitoring of treatment is used to indicate whether the treatment should be modified or continued as is.
  • 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.
  • 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.
  • 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.
  • 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.
  • signs and/or symptoms including dry eyes, dry mouth, headaches or migraines, muscle cramps, loss of appetite, seizure, stroke, dementia, gastrointestinal issues,
  • Some methods monitor at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or all of these signs and/or symptoms.
  • TTR amyloidosis 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
  • 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).
  • 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.
  • 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.
  • 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.
  • ECG electrocardiogram
  • 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.
  • 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.
  • a predetermined percentage of the peak less baseline e.g. 50%, 25% or 10%
  • administration of a further dose of antibody is administered.
  • 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.
  • TTR amyloid or pathogenic forms of TTR e.g, monomeric, misfolded, aggregated, or fibril forms of TTR
  • 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 am
  • 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.
  • levels of monomeric, misfolded, aggregated, or fibril forms of TTR in such subjects may be compared to those present in healthy subjects.
  • levels of TTR amyloid or pathogenic forms of TTR e.g., monomeric, misfolded, aggregated, or fibril forms of TTR
  • TTR amyloid or pathogenic forms of TTR e.g., monomeric, misfolded, aggregated, or fibril forms of TTR
  • 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.
  • Some such tests are sandwich immunoassays.
  • Some such immunoassays employ the Meso Scale Discovery (MSD) electrochemiluminescence platform (Meso Scale Diagnostics, Rockville, MD.)
  • MSD Meso Scale Discovery
  • Some such immunoassays use electrochemiluminescent labels on reporter antibodies, e.g., MSD Assays (Meso Scale Diagnostics, Rockville, MD.)
  • 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.
  • the labeled capture antibody can be 9D5 or 18C5 or a humanized, chimeric, or veneered variant thereof.
  • Some assays employ a labeled reporter antibody.
  • 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.
  • 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).
  • the antibody that binds TTR with no conformational specificity can be a polyclonal antibody.
  • the polyclonal antibody is a polyclonal rabbit anti-human prealbumin (Cat. No. A000202-2, Dako, Agilent Technologies, Inc, Santa Clara, CA).
  • the polyclonal rabbit anti-TTR antibody is Sigma, Catalog No. HPA002550 (Sigma-Aldrich, St. Louis, MO),
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • the capture antibody binds a first misfolded TTR molecule in the multimeric misfolded TTR
  • 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.
  • 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.
  • 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.
  • TTR transthyretin
  • 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.
  • misfolded 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.
  • the capture antibody binds a first misfolded TTR molecule in the multimeric misfolded TTR
  • 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.
  • 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.
  • a ratio of multimeric misfolded TTR to all misfolded TTR is calculated.
  • 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.
  • 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 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.
  • 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.
  • a reagent such as antibody that binds to monomeric, misfolded, aggregated, or fibril forms of TTR in the subject.
  • Such antibodies typically bind to an epitope within residues 89-97 or 101-109 of TTR.
  • the clearing response can be avoided by using antibody fragments lacking a full length constant region, such as Fabs.
  • the same antibody can serve as both a treatment and diagnostic reagent.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • agents such as a TTR tetramer stabilizer, an antisense oligonucleotide based therapeutic, an RNA interference (RNAi) based therapeutic or doxycycline plus tauroursodeoxycholic acid.
  • 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).
  • induction i.e., that reduces the level of amount of misfolded TTR, multimeric misfolded TTR, transthyretin deposits, or anti-TTR antibody binding
  • 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.
  • 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.
  • RNAi RNA interference
  • 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.
  • 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.
  • 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.
  • agents such as a TTR tetramer stabilizer, an antisense oligonucleotide based therapeutic, an RNA interference (RNAi) based therapeutic or doxycycline plus tauroursodeoxycholic acid.
  • 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.
  • 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.
  • 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.
  • an antibody is referred to in the present assay as being a test antibody because it is being tested for its target engagement.
  • 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.
  • detection of the reporter antibody as part of a sandwich provides an indication of presence and amount of misfolded TTR in a sample.
  • 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.
  • the biological sample is a plasma sample.
  • 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.
  • the test antibody is 14G8, and the assay is performed with a 9D5 or 18C5 capture antibody and a polyclonal anti-TTR reporter antibody.
  • the assays disclosed herein can be used to measure pharmocodynamic effects of therapies targeting misfolded forms of TTR.
  • 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.
  • 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.
  • the target of the test antibody is an epitope within residues 89-97 of TTR.
  • the target of the test antibody is an epitope within residues 101- 109 of TTR.
  • 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.
  • a transthyretin amyloidosis from a non- TTR amyloidosis, e.g. amyloid light-chain (AL) amyloidosis, also known as primary systemic amyloidosis.
  • AL amyloid light-chain
  • 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.
  • ATTR-PSS ATTR amyloidosis symptom survey
  • ARC Amyloidosis Research Consortium
  • 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.
  • ICF informed consent form
  • the study was approved by the New England Independent Review Board (NEIRB).
  • the moderator conducted the cognitive debriefing (CD) of the ATTR-PSS.
  • CD cognitive debriefing
  • 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.
  • 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.
  • 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.
  • Table 4 contains the demographics and disease characteristics of the seven focus group patients:
  • FIG. 1 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.
  • 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.
  • Shortness of Breath 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.”
  • 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 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.”
  • 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.
  • 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.
  • ATTR-PSS ATTR Amyloidosis Patient Symptom Survey
  • Example 2 Content Validation of the ATTR Amyloidosis Patient Symptom Survey: Findings from patient and clinician cognitive debriefing interviews
  • the ATTR Patient Symptom Survey 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.
  • ASG Amyloidosis Support Groups
  • 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.
  • ATTR-PSS 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
  • 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.’
  • 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.
  • 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.
  • 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
  • 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.
  • ATTR-PSS Amyloid transthyretin amyloidosis patient symptom survey
  • ATTRm hereditary ATTR amyloidosis
  • ATTRwt Wild type ATTR amyloidosis
  • 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
  • Fatigue is a big one.
  • the heart failure symptoms Anything with shortness of breath, dizziness, edema.
  • the pain ones are very important.
  • ATTR-PSS Amyloid transthyretin amyloidosis patient symptom survey
  • CM cardiomyopathy
  • ATTRm hereditary ATTR amyloidosis
  • ATTRwt wild type ATTR amyloidosis
  • PN peripheral neuropathy
  • 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.
  • 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.

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Abstract

L'invention concerne des procédés de surveillance ou de diagnostic de l'amyloïdose de la TTR à partir de signes et/ou de symptômes d'un sujet. L'invention est basée en partie sur l'identification de plusieurs signes et/ou symptômes non reconnus jusqu'à présent sur la base d'un groupe d'étude de patient et d'un groupe d'étude de clinicien. Des changements de signes et/ou de symptômes peuvent être utilisés, par exemple, pour initier ou modifier des régimes de traitement.
PCT/US2021/018632 2020-02-20 2021-02-18 Surveillance de l'amyloïdose de la transthyrétine WO2021168156A1 (fr)

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US11267877B2 (en) 2017-10-06 2022-03-08 Prothena Biosciences Limited Anti-transthyretin antibodies
US11267878B2 (en) 2015-01-28 2022-03-08 Neotope Neuroscience Limited Anti-transthyretin antibodies
CN114509572A (zh) * 2022-02-28 2022-05-17 大连润生康泰医学检验实验室有限公司 一种淀粉样变性药物药效定量评价方法
US11629185B2 (en) 2015-01-28 2023-04-18 Novo Nordisk A/S Anti-transthyretin antibodies
US11873332B2 (en) 2017-11-29 2024-01-16 Novo Nordisk A/S Lyophilized formulation of a monoclonal antibody against transthyretin
US11912759B2 (en) 2015-01-28 2024-02-27 Novo Nordisk A/S Anti-transthyretin antibodies

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11267878B2 (en) 2015-01-28 2022-03-08 Neotope Neuroscience Limited Anti-transthyretin antibodies
US11629185B2 (en) 2015-01-28 2023-04-18 Novo Nordisk A/S Anti-transthyretin antibodies
US11912759B2 (en) 2015-01-28 2024-02-27 Novo Nordisk A/S Anti-transthyretin antibodies
US11267877B2 (en) 2017-10-06 2022-03-08 Prothena Biosciences Limited Anti-transthyretin antibodies
US11873332B2 (en) 2017-11-29 2024-01-16 Novo Nordisk A/S Lyophilized formulation of a monoclonal antibody against transthyretin
CN114509572A (zh) * 2022-02-28 2022-05-17 大连润生康泰医学检验实验室有限公司 一种淀粉样变性药物药效定量评价方法

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