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WO2024130165A1 - Inhibiteurs de l'angptl3 pour la réduction des triglycérides dans le syndrome de chylomicronémie multifactorielle - Google Patents

Inhibiteurs de l'angptl3 pour la réduction des triglycérides dans le syndrome de chylomicronémie multifactorielle Download PDF

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Publication number
WO2024130165A1
WO2024130165A1 PCT/US2023/084357 US2023084357W WO2024130165A1 WO 2024130165 A1 WO2024130165 A1 WO 2024130165A1 US 2023084357 W US2023084357 W US 2023084357W WO 2024130165 A1 WO2024130165 A1 WO 2024130165A1
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antibody
angptl3
subject
seq
hangptl3
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Robert C. Pordy
Manish P. PONDA
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present disclosure is related to the use of ANGPTL3 inhibitors for reducing triglycerides in subjects having multifactorial chylomicronemia syndrome (MCS).
  • MCS multifactorial chylomicronemia syndrome
  • sHTG severe hypertriglyceridemia
  • MCS multifactorial chylomicronemia syndrome
  • LPL lipoprotein lipase
  • APO apolipoprotein
  • GPIHBP1 glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1
  • LMF1 lipase maturation factor 1
  • Angiopoietin-like 3 (ANGPTL3) is an important regulator of lipoprotein metabolism, acting as an inhibitor of LPL and endothelial lipase (EL) (Musunuru, et al., 2010, NEJM, 363:2220-2227; Adam, et al., 2020, J Lipid Res, 61 :1271-1286).
  • EL endothelial lipase
  • Individuals with LOF variants in ANGPTL3 have significantly reduced triglycerides, suggesting that it could be a therapeutic target for lowering triglycerides by increasing LPL activity (Dewey, et al., 2017.
  • Evinacumab is a fully human monoclonal antibody that inhibits ANGPTL3 (Adam, et al., 2020, J Lipid Res, 61 :1271-1286; Gaudet, et al., 2017, NEJM, 377 29Q-297', Raal, et al., 2020, NEJM, 383:711-720), and prior studies have assessed its efficacy and safety in patients with hypertriglyceridemia (Ahmad, et al., 2019, Circulation, 140:470-486; Ahmad, et al., 2021 , J Am Coll Cardiol, 78:193-195).
  • Antibodies to ANGPTL3 are disclosed in, for example, US 9,018,356, W02008/073300, and US 7,935,796.
  • the disclosure provides methods for treating or alleviating a symptom of severe hypertriglyceridemia in a subject, comprising administering an inhibitor of AngPTL3 to the subject.
  • severe hypertriglyceridemia is defined as a triglyceride (TG) concentration > 500 mg/dL (> 5.7 mmol/L) or a TG level >880 mg/dL (>10 mmol/L).
  • severe hypertriglyceridemia (sHTG) is defined as a triglyceride (TG) concentration > 1000 mg/dL.
  • the symptom is selected from the group consisting of elevated triglycerides, inflammation of the pancreas, abdominal pain, enlarged liver, enlarged spleen, xanthomas, nerve damage (e.g., loss of feeling in feet or legs, memory loss), elevated deposition of atherogenic triglyceride-rich lipoproteins, and elevated deposition of triglyceride-rich lipoproteins in parenchymal tissues.
  • the symptom comprises elevated trigycerides, as described above.
  • the symptom is selected from the group consisting of elevated triglycerides, inflammation of the pancreas, abdominal pain, enlarged liver, enlarged spleen, xanthomas, nerve damage (e.g., loss of feeling in feet or legs, memory loss), elevated deposition of atherogenic triglyceride-rich lipoproteins, and elevated deposition of triglyceride-rich lipoproteins in parenchymal tissues.
  • the symptom comprises elevated trigycerides, as described above.
  • the disclosure provides methods of reducing triglycerides in a subject with multifactorial chylomicronemia syndrome (MCS), comprising selecting a subject with MCS that exhibits elevated triglycerides and optionally at least one other symptom of MCS, and administering an inhibitor of ANGPTL3 to the subject.
  • MCS multifactorial chylomicronemia syndrome
  • the disclosure provides methods of reducing triglycerides in a subject with multifactorial chylomicronemia syndrome (MCS), comprising selecting a subject with MCS that exhibits elevated triglycerides and/or at least one other symptom of MCS, and administering an inhibitor of ANGPTL3 to the subject.
  • MCS is defined on the basis of a triglyceride (TG) concentration > 500 mg/dL (> 5.7 mmol/L) or a TG level >880 mg/dL (>10 mmol/L).
  • the at least one other symptom is selected from the group consisting of inflammation of the pancreas, abdominal pain, enlarged liver, enlarged spleen, xanthomas, nerve damage (e.g., loss of feeling in feet or legs, memory loss), elevated deposition of atherogenic triglyceride-rich lipoproteins, and elevated deposition of triglyceride-rich lipoproteins in parenchymal tissues.
  • the inhibitor of ANGPTL3 is an antibody or antigen-binding fragment thereof that specifically binds ANGPTL3.
  • the anti-ANGPTL3 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) of a heavy chain variable region (HCVR) / light chain variable region (LCVR) pair comprising SEQ ID NOs:66/74.
  • the anti-ANGPTL3 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, HCDR3) comprising SEQ ID NOs:68, 70, 72, respectively, and three light chain complementarity determining regions (LCDR1 , LCDR2, LCDR3) comprising SEQ ID NOs:76, 78, and 80, respectively.
  • the anti-ANGPTL3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising SEQ ID NO:66 and a light chain variable region (LCVR) comprising SEQ ID NO: 74.
  • the ANGPTL3 inhibitor is evinacumab or a bioequivalent thereof.
  • the ANGPTL3 inhibitor is administered to the subject at a dosage of about 15 mg/kg. In yet another embodiment, the ANGPTL3 inhibitor is administered to the subject at a dosage of about 15 mg/kg Q4W.
  • the administration of the ANGPTL3 inhibitor to the subject results in at least one of the parameter changes selected from the group consisting of: i) at least a 60% decrease in fasting triglycerides in the subject; ii) at least a 30% decrease in total cholesterol in the subject; iii) at least a 30% decrease in non-HDL-C lipoproteins in the subject; and iv) at least a 60% decrease in remnant cholesterol in the subject .
  • the administration of the ANGPTL3 inhibitor to the subject results in at least one of the parameter changes selected from the group consisting of: i) at least an 80% decrease in fasting triglycerides in the subject; ii) at least a 33% decrease in total cholesterol in the subject; iii) at least a 37% decrease in non-HDL-C lipoproteins in the subject; and iv) at least a 75% decrease in remnant cholesterol in the subject.
  • the ANGPTL3 inhibitors are fully human monoclonal antibodies (mAbs) and antigen-binding fragments thereof that specifically bind and neutralize, inhibit, block, abrogate, reduce or interfere with, at least one activity of ANGTPL3, in particular, human ANGPTL3 (SEQ ID NO:161).
  • the activity of ANGPTL3 that can be neutralized, inhibited, blocked, abrogated, reduced or interfered with, by the antibodies or fragments thereof of the disclosure includes, but not by the way of limitation, inhibition of LPL activity, induction of angiogenesis, and the like.
  • an antibody or fragment thereof used in the present disclosure can neutralize, inhibit, block, abrogate, reduce or interfere with, an activity of hANGPTL3 by binding to an epitope of hANGPTL3 that is directly involved in the targeted activity of hANGPTL3.
  • an antibody or fragment thereof of the disclosure can neutralize, inhibit, block, abrogate, reduce or interfere with, an activity of hANGPTL3 by binding to an epitope of hANGPTL3 that is not directly involved in the targeted activity of hANGPTL3, but the antibody or fragment binding thereto sterically or conformationally inhibits, blocks, abrogates, reduces or interferes with, the targeted activity of hANGPTL3.
  • an antibody or fragment thereof used in the disclosure binds to an epitope of hANGPTL3 that is not directly involved in the targeted activity (e.g., inhibiting LPL activity, inducing angiogenesis, and the like) of hANGPTL3 (/.e., a nonblocking antibody), but the antibody or fragment binding thereto results in the enhancement of the clearance of hANGPTL3 from the circulation, compared to the clearance of hANGPTL3 in the absence of the antibody or fragment thereof, thereby indirectly inhibiting, blocking, abrogating, reducing or interfering with, an activity of hANGPTL3. Clearance of hANGPTL3 from the circulation can be particularly enhanced by combining two or more different non-blocking antibodies that do not compete with one another for specific binding to hANGPTL3.
  • the antibodies (Abs) can be full-length (for example, an IgG 1 or lgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab’) 2 or scFv fragment), and may be modified to affect functionality, e.g., to eliminate residual effector functions (Reddy, et al., 2000, J. Immunol., 164:1925-1933).
  • the ANGPTL3 inhibitor is an antibody or antigen-binding fragment of an antibody comprising a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NO:2, 18, 34, 50, 66, 82, 98, 114, 130, 146 and 180, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • the antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO:2, 18, 34, 66, 82, 114, and 180.
  • the antibody or an antigen-binding fragment thereof comprises a HCVR having an amino acid sequence of SEQ ID NO:66.
  • the ANGPTL3 inhibitor is an antibody or antigen-binding fragment of an antibody that comprises a light chain variable region (LCVR) selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154 and 188, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • LCVR light chain variable region
  • the antibody or antigen-binding portion of an antibody comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 74, 90, 122 and 188. In yet another embodiment, the antibody or antigen-binding portion of an antibody comprises a LCVR having an amino acid sequence of SEQ ID NO: 74.
  • the antibody or fragment thereof comprises a HCVR and LCVR sequence pair (HCVR/LCVR) selected from the group consisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154 and 180/188.
  • the antibody or fragment thereof comprises a HCVR and LCVR sequence pair selected from the group consisting of SEQ ID NO:2/10, 18/26, 34/42, 66/74, 82/90, 114/122 and 180/188.
  • the antibody or fragment thereof comprises a HCVR and LCVR sequence pair of SEQ ID NO:66/74.
  • the ANGPTL3 inhibitor is an antibody or antigen-binding fragment of an antibody comprising a heavy chain complementarity determining region 3 (HCDR3) amino acid sequence selected from the group consisting of SEQ ID NO:8, 24, 40, 56, 72, 88, 104, 120, 136, 152 and 186, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR3 (LCDR3) amino acid sequence selected from the group consisting of SEQ ID NO:16, 32, 48, 64, 80, 96, 112, 128, 144, 160 and 194, or substantially similar sequences thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • HCDR3 heavy chain complementarity determining region 3
  • the antibody or fragment thereof comprises a HCDR3/LCDR3 amino acid sequence pair comprising SEQ ID NO:8/16, 24/32, 40/48, 56/64, 72/80, 88/96, 104/112, 120/128, 136/144, 152/160 or 186/194.
  • the antibody or fragment thereof comprises a HCDR3/LCDR3 amino acid sequence pair comprising SEQ ID NO:8/16, 24/32, 40/48, 72/80, 88/96, 120/128 or 186/194.
  • the antibody or fragment thereof comprises a HCDR3/LCDR3 amino acid sequence pair comprising SEQ ID NO:72/80.
  • the antibody or fragment thereof further comprises a heavy chain CDR1 (HCDR1) amino acid sequence selected from the group consisting of SEQ ID NO:4, 20, 36, 52, 68, 84, 100, 116, 132, 148 and 182, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a heavy chain CDR2 (HCDR2) amino acid sequence selected from the group consisting of SEQ ID NO:6, 22, 38, 54, 70, 86, 102, 118, 134, 150 and 184, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and optionally further comprises a light chain CDR1 (LCDR1) amino acid sequence selected from the group consisting of SEQ ID NO:12, 28, 44, 60, 76, 92, 108, 124, 140, 156 and 190, or a substantially similar sequence thereof having at least 90%, at least 95%, at least
  • the ANGPTL3 inhibitor is an antibody or antigen-binding fragment of an antibody comprising a HCDR1/HCDR2/HCDR3 combination selected from the group consisting of SEQ ID NO:4/6/8, 20/22/24, 36/38/40, 52/54/56, 68/70/72, 84/86/88, 100/102/104, 116/118/120, 132/134/136, 148/150/152 and 182/184/186; and/or a LCDR1/LCDR2/LCDR3 combination selected from the group consisting of SEQ ID NO:12/AAS/16, 28/KAS/32, 44/AAS/48, 60/KAS/64, 76/KAS/80, 92/TTS/96, 108/PAS/112, 124/TAS/128, 140/KVS/144, 156/ AA/160 and 190/KAS/194.
  • a HCDR1/HCDR2/HCDR3 combination selected from the group consisting of SEQ ID NO:4/6/8, 20/22/24
  • the heavy and light chain CDR amino acid sequences comprise a CDR sequence combination selected from the group consisting of SEQ ID NO:4/6/8/12/AAS/16, 20/22/24/28/KAS/32, 36/38/40/44/AAS/48, 52/54/56/60/KAS/64, 68/70/72/76/KAS/80, 84/86/88/92/TTS/96, 100/102/104/108/PAS/112, 116/118/120/124/TAS/128, 132/134/136/140/KVS/144, 148/150/152/156/VAA/160 and 182/184/186/190/KAS/194.
  • the heavy and light chain CDR amino acid sequences comprise a CDR sequence combination of SEQ ID NO: 4/6/8/12/AAS/16, 20/22/24/28/KAS/32, 36/38/40/44/AAS/48, 68/70/72/76/KAS/80, 84/86/88/92/TTS/96, 116/118/120/124/TAS/128 or 182/184/186/190/KAS/194.
  • the heavy and light chain CDR amino acid sequences comprise a CDR sequence combination of SEQ ID NQ:68/70/72/76/KAS/80.
  • the ANGPTL3 inhibitor is an antibody or antigen-binding fragment of an antibody that specifically binds hANGPTL3, wherein the antibody or fragment thereof comprises heavy and light chain CDR domains contained within HCVR/LCVR pairs selected from the group consisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154 and 180/188.
  • Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are known in the art and can be applied to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein.
  • the antibody or fragment thereof comprises CDR sequences contained within a HCVR and LCVR pair of SEQ ID NO: 2/10, 18/26, 34/42, 66/74, 82/90, 114/122 or 180/188. In another embodiment, the antibody or fragment thereof comprises CDR sequences contained within a HCVR and LCVR pair of SEQ ID NO:66/74.
  • the ANGPTL3 inhibitor is an antibody or antigen-binding fragment thereof that competes for specific binding to hANGPTL3 with an antibody or antigen-binding fragment comprising heavy and light chain CDR sequences contained in a HCVR/LCVR sequence pair of SEQ ID NQ:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154 or 180/188.
  • the antibody or antigen-binding fragment of the disclosure competes for specific binding to hANGPTL3 with an antibody or fragment thereof comprising a HCVR/LCVR sequence pair of SEQ ID NO:66/74.
  • the antibody or antigen-binding fragment used in the disclosure competes for specific binding to hANGPTL3 with an antibody or fragment thereof comprising a heavy and light chain CDR sequence combination selected from the group consisting of SEQ ID NOS 4/6/8/12/AAS/16, 20/22/24/28/KAS/32, 36/38/40/44/AAS/48, 52/54/56/60/KAS/64, 68/70/72/76/KAS/80, 84/86/88/92/TTS/96, 100/102/104/108/PAS/112,
  • the antibody or antigen-binding fragment thereof used in the disclosure competes for specific binding to hANGPTL3 with an antibody or fragment thereof comprising a heavy and light chain CDR sequence combination of SEQ ID NOS:68/70/72/76/KAS/80.
  • the ANGPTL3 inhibitor is an antibody or antigen-binding fragment thereof that binds the same epitope on hANGPTL3 that is recognized by an antibody or fragment thereof comprising heavy and light chain CDR sequences from a HCVR/LCVR sequence pair of SEQ ID NQ:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154 or 180/188.
  • the antibody or antigen-biding fragment used in the disclosure binds the same epitope on hANGPTL3 as that recognized by the antibody or fragment thereof comprising a HCVR/LCVR sequence pair of SEQ ID NO:66/74. In one embodiment, the antibody or fragment thereof used in the disclosure binds the same epitope on hANGPTL3 that is recognized by an antibody or fragment thereof comprising a heavy and light chain CDR sequence combination selected from the group consisting of SEQ ID NOS 4/6/8/12/AAS/16, 20/22/24/28/KAS/32, 36/38/40/44/AAS/48, 52/54/56/60/KAS/64, 68/70/72/76/KAS/80, 84/86/88/92/TTS/96, 100/102/104/108/PAS/112,
  • such an epitope is recognized by an antibody or fragment thereof comprising a heavy and light chain CDR sequence combination of SEQ ID NO:68/70/72/76/KAS/80.
  • the ANGPTL3 inhibitor is an isolated anti-hANGPTL3 antibody or antigen-binding fragment thereof that binds to an epitope situated within the N-terminal coiled-coil region at residues 17 to 209 of SEQ ID NO: 161 and neutralizes, inhibits, abrogates, reduces or interferes with, at least one activity of hANGPTL3.
  • the ANGPTL3 inhibitor is an isolated antibody or antigen-binding fragment of an antibody that specifically binds to an epitope situated within the N-terminal coiled-coil region of hANGPTL3 (SEQ ID NO:161) and neutralizes, inhibits, abrogates, reduces or interferes with, at least one activity of hANGPTL3, with the proviso that the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO: 170 (corresponds to residues Glu32 to Leu57 of hANGPTL3 of SEQ ID NO:161).
  • the antibody or fragment thereof used in the disclosure specifically binds to an epitope within residues 17 to 200, 17 to 100, 17 to 70, 17 to 65, 17 to 60, 17 to 57, or 17 to 50, of hANGPTL3 (SEQ ID NO:161), optionally with the proviso that the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO: 170.
  • the antibody or fragment thereof specifically binds to an epitope within residues 40 to 200, 40 to 100, 40 to 70, 50 to 200, 50 to 100, 50 to 70, 58 to 200, 58 to 100, 58 to 70, 58 to 68, or 61 to 66, of hANGPTL3 (SEQ ID NO:161), optionally with the proviso that the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NQ:170.
  • the antibody or antibody fragment binds an epitope which may involve more than one of the enumerated epitopes or residues within the N-terminal coiled-coil region of hANGPTL3, optionally with the proviso that the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO: 170.
  • the ANGPTL3 inhibitor is an antibody or fragment thereof comprising a HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 , 17, 33, 49, 65, 81, 97, 113, 129, 145 and 179, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof.
  • the antibody or fragment thereof comprises a HCVR encoded by a nucleic acid sequence of SEQ ID NO:1 , 17, 33, 65, 81, 113 or 179.
  • the antibody or fragment thereof comprises a HCVR encoded by a nucleic acid sequence of SEQ ID NO:65.
  • an antibody or antigen-binding fragment thereof used herein comprises a LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, 25, 41 , 57, 73, 89, 105, 121 , 137, 153 and 187, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof.
  • the antibody or fragment thereof comprises a LCVR encoded by a nucleic acid sequence of SEQ ID NO:9, 25, 41 , 73, 89, 121 or 187.
  • the antibody or fragment thereof comprises a LCVR encoded by a nucleic acid sequence of SEQ ID NO:73.
  • the ANGPTL3 inhibitor is an antibody or antigen-binding fragment thereof that comprises a HCVR and LCVR (HCVR/LCVR) sequence pair encoded by a nucleic acid sequence pair selected from the group consisting of SEQ ID NO:1/9, 17/25, 33/41 , 49/57, 65/73, 81/89, 97/105, 113/121 , 129/137, 145/153 and 179/187.
  • HCVR/LCVR HCVR/LCVR
  • the antibody or fragment thereof comprises a HCVR/LCVR sequence pair encoded by a nucleic acid sequence pair of SEQ ID NO:1/9, 17/25, 33/41 , 65/73, 81/89, 113/121 or 179/187. In another embodiment, the antibody or fragment thereof comprises a HCVR/LCVR sequence pair encoded by a nucleic acid sequence pair of SEQ ID NO:65/73.
  • the ANGPTL3 inhibitor is an antibody or antigen-binding fragment of an antibody comprising a HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:7, 23, 39, 55, 71 , 87, 103, 119, 135, 151 and 185, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof; and a LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:15, 31 , 47, 63, 79, 95, 111 , 127, 143, 159 and 193, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof.
  • the antibody or fragment thereof comprises a HCDR3 and LCDR3 sequence pair encoded by the nucleic acid sequence pair selected from the group consisting of SEQ ID NO:7/15, 23/31 , 39/47, 55/63, 71/79, 87/95, 103/111 , 119/127, 135/143, 151/159 and 185/193.
  • the antibody or fragment thereof comprises a HCDR3 and LCDR3 sequence pair encoded by the nucleic acid sequence pair of SEQ ID NO:7/15, 23/31 , 39/47, 71/79, 87/95, 119/127 or 185/193.
  • the antibody or fragment thereof comprises a HCDR3 and LCDR3 sequence pair encoded by the nucleic acid sequence pair of SEQ ID NO:71/79.
  • the ANGPTL3 inhibitor is an antibody or antigen-binding fragment thereof that further comprises a HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 19, 35, 51 , 67, 83, 99, 115, 131 , 147 and 181 , or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof; and a HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:5, 21 , 37, 53, 69, 85, 101 , 117, 133, 149 and 183, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof; and optionally further comprises a LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:11 , 27, 43,
  • the ANGPTL3 inhibitor is an antibody or antigen-binding fragment of an antibody comprising a HCDR1/HCDR2/HCDR3 combination encoded by a nucleotide sequence combination selected from the group consisting of SEQ ID NO:3/5/7, 19/21/23, 35/37/39, 51/53/55, 67/69/71, 83/85/87, 99/101/103, 115/117/119, 131/133/135, 147/149/151 and 181/183/185; and/or a LCDR1/LCDR2/LCDR3 combination encoded by a nucleotide sequence combination selected from the group consisting of SEQ ID NO:3/5/7, 19/21/23, 35/37/39, 51/53/55, 67/69/71, 83/85/87, 99/101/103, 115/117/119, 131/133/135, 147/149/151 and 181/183/185; and/or a LCDR1/LCDR2/LCDR3 combination encoded by a nucleotide sequence combination selected
  • the antibody or fragment thereof comprises heavy and light chain CDR sequences encoded by a nucleotide sequence combination of SEQ ID NO:67/69/71/75/aaggcgtct/79.
  • the ANGPTL3 inhibitor is a human anti-ANGPTL3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) encoded by nucleotide sequence segments derived from V , D and J germline sequences, and a light chain variable region (LCVR) encoded by nucleotide sequence segments derived from V K and J K germline sequences, wherein the HCVR and the LCVR are encoded by nucleotide sequence segments derived from a germline gene combination selected from the group consisting of: (i) VH3-43, DH3-3, JH3, VK1-5 and JK2; (ii) VH3-1 1 , DH1 -1 , JH4, VK1-39 and JK4; (iii) V H 3-30, D H 1-7, J H 6, V K 1-5 and J K 1; (iv) V H 3-30, D H 1-26, J H 6, V K 1-12
  • the ANGPTL3 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to hANGPTL3 with an equilibrium dissociation constant (K D ) of about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less, as measured by surface plasmon resonance assay (for example, BIACORETM).
  • K D equilibrium dissociation constant
  • the antibody of the disclosure exhibits a K D of about 800 pM or less, about 700 pM or less; about 600 pM or less; about 500 pM or less; about 400 pM or less; about 300 pM or less; about 200 pM or less; about 100 pM or less; or about 50 pM or less.
  • the ANGPTL3 inhibitor is an anti-hANGPTL3 antibody or antigen-binding fragment thereof that binds hANGPTL3 protein of SEQ ID NO: 161 , but does not cross-react with a related protein, such as a human angiopoietin-like protein 4 (hANGPTL4; SEQ ID NO:164), as determined by, for example, ELISA, surface plasmon resonance assay, or LUMINEX® XMAP® Technology, as described herein.
  • hANGPTL4 human angiopoietin-like protein 4
  • ANGPTL4 is another secreted protein that is known to reduce LPL activity and has an N-terminal coiled-coil region and a C-terminal fibrinogen- like domain (Ge, et al., 2004, J Biol Chem, 279:2038-2045; Yau, et al., 2009, J Biol Chem, 284:11942-11952).
  • the disclosure provides an anti-hANGPTL3 antibody or antigen binding fragment thereof that binds a hANGPTL3 protein and crossreacts with a hANGPTL4 protein.
  • the binding affinity of the hANGPTL3 antibody or fragment thereof to hANGPTL4 protein is about 75% or less, or about 50% or less, of the binding affinity of the antibody or fragment to the hANGPTL3 protein.
  • the ANGPTL3 inhibitor is an anti-hANGPTL3 antibody or antigen binding fragment thereof that does not cross-react with mouse ANGPTL3 (mANGPTL3; SEQ ID NO:163), or rat ANGPTL3 (rANGPTL3; SEQ ID NO:175), but does cross-react with cynomolgus monkey (Macaca fascicularis) ANGPTL3 (MfANGPTL3), for example, with the N-terminal 17-170 residues of SEQ ID NO: 177 (a partial amino acid sequence of MfANGPTL3).
  • the ANGPTL3 inhibitor is an anti-hANGPTL3 antibody or fragment thereof that cross-reacts with MfANGPTL3, mANGPTL3 and rANGTPL3.
  • the ANGPTL3 inhibitor is an anti-hANGPTL3 antibody having a modified glycosylation pattern.
  • modification to remove undesirable glycosylation sites may be useful, or e.g., removal of a fucose moiety to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield, et al., (2002) JBC 277:26733).
  • ADCC antibody dependent cellular cytotoxicity
  • removal of N- glycosylation site may reduce undesirable immune reactions against the therapeutic antibodies, or increase affinities of the antibodies.
  • modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).
  • a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds hANGPTL3 and a pharmaceutically acceptable carrier is administered to a subject
  • the pharmaceutical composition comprises one or more anti-ANGPTL3 antibodies or fragments thereof described herein, which do not crosscompete with one another, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can contain two or more non-blocking antibodies, which do not compete with one another for specific binding to hANGPTL3 and are effective in clearing hANGPTL3 from the circulation.
  • Suitable combinations of nonblocking antibodies include, but are not limited to, a combination of antibodies comprising HCVR and LCVR sequence pairs (HCVR/LCVR) of: (i) SEQ ID NO:82/90 and 180/188, respectively; (ii) SEQ ID NO:114/122 and 180/188, respectively; (iii) SEQ ID NQ:82/90 and 18/26, respectively; or (iv) SEQ ID NO:114/122 and 18/26, respectively.
  • HCVR/LCVR HCVR sequence pairs
  • the composition is a combination of an antibody or antigen-binding fragment thereof of as described herein, and a second therapeutic agent.
  • the second therapeutic agent may be one or more of any agent such as (1) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, such as cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, and the like; (2) inhibitors of cholesterol uptake and/or bile acid re-absorption; (3) niacin, which increases lipoprotein catabolism; (4) fibrates or amphipathic carboxylic acids, which reduce low-density lipoprotein (LDL) level, improve high-density lipoprotein (HDL) and TG levels, and reduce the number of non-fatal heart attacks; and (5) activators of the LXR transcription factor that plays
  • HMG-CoA 3-hydroxy-3-methylgluta
  • the second therapeutic agent can be one or more other inhibitors of ANGPTL3 as well as inhibitors of other molecules, such as ANGPTL4, ANGPTL5, ANGPTL6 and proprotein convertase subtilisin/kexin type 9 (PCSK9), which are involved in lipid metabolism, in particular, cholesterol and/or triglyceride homeostasis.
  • Inhibitors of these molecules include small molecules and antibodies that specifically bind to these molecules and block their activity.
  • the second therapeutic agent may be one or more anticancer agents, such as chemotherapeutic agents, anti-angiogenic agents, growth inhibitory agents, cytotoxic agents, apoptotic agents, and other agents well known in the art to treat cancer or other proliferative diseases or disorders, as well as other therapeutic agents, such as analgesics, anti-inflammatory agents, including non-steroidal antiinflammatory drugs (NSAIDS), such as Cox-2 inhibitors, and the like, so as to ameliorate and/or reduce the symptoms accompanying the underlying cancer/tumor.
  • anticancer agents such as chemotherapeutic agents, anti-angiogenic agents, growth inhibitory agents, cytotoxic agents, apoptotic agents, and other agents well known in the art to treat cancer or other proliferative diseases or disorders, as well as other therapeutic agents, such as analgesics, anti-inflammatory agents, including non-steroidal antiinflammatory drugs (NSAIDS), such as Cox-2 inhibitors, and the like, so as to ameliorate and/or reduce the symptoms accompanying the underlying
  • the methods of the disclosure can also prevent or treat diseases or disorders associated with or resulting from severe hypertriglyceridemia, including, but not limited to, cardiovascular diseases or disorders, such as atherosclerosis, aneurysm, hypertension, angina, stroke, cerebrovascular diseases, congestive heart failure, coronary artery diseases, myocardial infarction, peripheral vascular diseases, and the like; acute pancreatitis; nonalcoholic steatohepatitis (NASH); blood sugar disorders, such as diabetes; obesity, and the like.
  • cardiovascular diseases or disorders such as atherosclerosis, aneurysm, hypertension, angina, stroke, cerebrovascular diseases, congestive heart failure, coronary artery diseases, myocardial infarction, peripheral vascular diseases, and the like
  • acute pancreatitis nonalcoholic steatohepatitis (NASH)
  • NASH nonalcoholic steatohepatitis
  • blood sugar disorders such as diabetes; obesity, and the like.
  • Figures 1A-1C show Median (Q1 to Q3) percent change in fasting triglycerides from baseline to week 12 by cohort in the DBTP (exploratory endpoints): Figure 1A - Cohort 1 , Figure 1B - Cohort 2, and Figure 1C - Cohort 3.
  • Figure 2 provides a schematic depiction of the Study design for Example 3.
  • human angiopoietin-like protein 3 or "hANGPTL3”, as used herein, refers to ANGPTL3 having the nucleic acid sequence shown in SEQ ID NO:162 and the amino acid sequence of SEQ ID NO: 161 , or a biologically active fragment thereof.
  • antibody as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (HCVR) and a heavy chain constant region (CH; comprised of domains CH1 , CH2 and CH3).
  • HCVR heavy chain variable region
  • CH heavy chain constant region
  • Each light chain is comprised of a light chain variable region (LCVR) and a light chain constant region (CL).
  • the HCVR and LCVR can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each HCVR and LCVR is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1 , FR2, CDR2, FR3, CDR3, and FR4.
  • CDR residues not contacting antigen can be identified based on previous studies (for example, residues H60-H65 in CDRH2 are often not required), from regions of Kabat CDRs lying outside Chothia CDRs, by molecular modeling and/or empirically. If a CDR or residue(s) thereof is omitted, it is usually substituted with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human mAbs of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • human antibody as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse), have been grafted onto human FR sequences.
  • the fully-human anti-hANGPTL3 antibodies used herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
  • the present disclosure uses antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences described herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residues(s) (such sequence changes are referred to herein collectively as “germline mutations”).
  • germline mutations such sequence changes are referred to herein collectively as “germline mutations”.
  • all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived.
  • only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1 , CDR2 or CDR3.
  • one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (/.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).
  • the antibodies used herein may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residues of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
  • antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc.
  • Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the methods of present disclosure.
  • the present disclosure also uses anti-ANGPTL3 antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions.
  • the present disclosure uses anti- ANGPTL3 antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 2 or 1 , conservative amino acid substitution(s) relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.
  • a HCVR comprises the amino acid sequence of SEQ ID NO:487 with 10 or fewer conservative amino acid substitutions therein.
  • a HCVR comprises the amino acid sequence of SEQ ID NO:487 with 8 or fewer conservative amino acid substitutions therein. In another embodiment, a HCVR comprises the amino acid sequence of SEQ ID NO:487 with 6 or fewer conservative amino acid substitutions therein. In another embodiment, a HCVR comprises the amino acid sequence of SEQ ID NO:487 with 4 or fewer conservative amino acid substitutions therein. In yet another embodiment, a HCVR comprises the amino acid sequence of SEQ ID NO:487 with 2 or 1 conservative amino acid substitution(s) therein. In one embodiment, a LCVR comprises the amino acid sequence of SEQ ID NO:44 with 10 or fewer conservative amino acid substitutions therein.
  • a LCVR comprises the amino acid sequence of SEQ ID NO:44 with 8 or fewer conservative amino acid substitutions therein. In another embodiment, a LCVR comprises the amino acid sequence of SEQ ID NO:44 with 6 or fewer conservative amino acid substitutions therein. In another embodiment, a LCVR comprises the amino acid sequence of SEQ ID NO:44 with 4 or fewer conservative amino acid substitutions therein. In yet another embodiment, a LCVR comprises the amino acid sequence of SEQ ID NO:44 with 2 or 1 conservative amino acid substitution(s) therein.
  • antibody shall be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (/.e., “full antibody molecules”) as well as antigen-binding fragments thereof.
  • antibody portion of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigenbinding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-display antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.
  • SMIPs small modular immunopharmaceuticals
  • An antigen-binding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the V H and L domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain V H - V H , H- L or L-V L dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric V H or V L domain.
  • an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) V H - C H 1 ; (ii) VH-C H 2; (iii) V H -C H 3; (iv) VH-C H 1-C H 2; (V) V H -CH1 -C H 2-CH3; (vi) V H -C H 2-C H 3; (vii) VH-CL; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) L -C H 1-CH2; (xii) V L -CH1-C H 2-C H 3; (xiii) V L - CH2-CH3; and (xiv) VL-CL.
  • variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
  • a hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
  • an antigen-binding fragment of an antibody used in the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric H or L domain (e.g., by disulfide bond(s)).
  • antigen-binding fragments may be monospecific or multispecific (e.g., bispecific).
  • a multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.
  • Any multispecific antibody format, including the exemplary bispecific antibody formats described herein, may be adapted for use in the context of an antigen-binding fragment of an antibody used in the present disclosure using routine techniques available in the art.
  • antibody or antibody fragments used in the disclosure may be conjugated to a therapeutic moiety (“immunoconjugate”), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope.
  • a therapeutic moiety such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope.
  • the term "specifically binds," or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant (K D ) of about 1x10’ 6 M or less (/.e., a smaller K D denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.
  • an isolated antibody that specifically binds hANGPTL3 may, however, exhibit cross-reactivity to other antigens, such as ANGPTL3 molecules from other species, for example, cynomolgus monkey ANGPTL3, mouse ANGPTL3, rat ANGPTL3, and/or hANGPTL4 having the amino acid sequence of SEQ ID NO: 164.
  • multi-specific antibodies e.g., bispecifics
  • that bind to hANGPTL3 and one or more additional antigens are nonetheless considered antibodies that “specifically bind” hANGPTL3, as used herein.
  • high affinity antibody refers to those antibodies having a binding affinity to hANGPTL3, expressed as KD, of about 2x1 O' 9 M or less, about 1.5x1 O’ 9 M or less, about 1x10’ 9 M or less, about 0.5x10’ 9 M or less, about 0.25x10’ 9 M or less, about 1x1 O’ 10 M or less, or about 0.5x1 O’ 10 M or less, as measured by surface plasmon resonance, e.g., BIACORETM or solution-affinity ELISA.
  • KD is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.
  • slow off rate an antibody that dissociates from hANGPTL3 with a rate constant of 4 x 10 -3 s -1 or less, 3 x 10 -3 s -1 or less, 2 x 10’ 3 S’ 1 or less, 1 x 10- S’ 1 or less, 1 x 10’ 4 S’ 1 or less, as determined by surface plasmon resonance, e.g., BIACORETM.
  • k a intrinsic affinity constant
  • an "isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other mAbs having different antigenic specificities (e.g., an isolated antibody that specifically binds hANGPTL3 is substantially free of mAbs that specifically bind antigens other than hANGPTL3).
  • An isolated antibody that specifically binds hANGPTL3 may, however, have cross-reactivity to other antigens, such as ANGPTL3 molecules from other species, such as cynomolgus monkey, mouse, rat, and/or other related proteins, such as human ANGPTL4.
  • a “neutralizing”, “blocking” or “abrogating” antibody, as used herein (or an antibody that “neutralizes”, “blocks” or “abrogates” ANGPTL3 activity), is intended to refer to an antibody whose binding to ANGPTL3 results in direct inhibition of at least one biological activity of ANGPTL3, as assessed by standard in vitro assays known in the art (for example, see Examples below).
  • the terms, “neutralize”, “inhibit”, “block” and “abrogate”, may be used herein interchangeably.
  • non-blocking antibody refers to an antibody whose binding to ANGPTL3 does not directly block a targeted activity of ANGPTL3 as assessed by standard in vitro assays, but yet may be an “interfering” antibody whose binding to ANGPTL3 results in indirect inhibition, reduction, attenuation, or other interference, of at least one biological activity of ANGPTL3 in vivo, e.g., by enhancing the clearance of ANGPTL3 from the circulation. Clearance of ANGPTL3 from the circulation can be particularly enhanced by a combination of at least two non-blocking antibodies.
  • the neutralization, inhibition, abrogation, reduction, attenuation or interference, of a biological activity of ANGPTL3 can be assessed by measuring one or more indicators of ANGPTL3 biological activity by one or more of several standard in vitro or in vivo assays known in the art (also see Examples below).
  • surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORETM system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).
  • epipe is a region of an antigen that is bound by an antibody.
  • Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
  • nucleic acid or fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well- known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below.
  • the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • a "conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, (1994), Methods Mol. Biol., 24: 307- 331.
  • Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
  • a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet, et al., (1992), Science, 256: 144345.
  • a "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
  • Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1.
  • FASTA e.g., FASTA2 and FASTA3
  • FASTA2 and FASTA3 provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra).
  • Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul, et al., (1990), J. Mol. Biol., 215: 403 410 and (1997), Nucleic Acids Res., 25:3389 402.
  • terapéuticaally effective amount is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, the age and the size of a subject treated, the route of administration, and the like, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd, (1999), The Art, Science and Technology of Pharmaceutical Compounding).
  • the antibodies used in the instant disclosure possess high affinities, typically possessing KD of from about 10 -12 M through about 10 -9 M, when measured by binding to antigen either immobilized on solid phase or in solution phase.
  • the mouse constant regions are replaced with desired human constant regions, for example, wildtype lgG1 or lgG4, or modified lgG1 or lgG4, to generate the fully human antibodies of the disclosure. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics of the antibodies reside in the variable region.
  • epitope refers to a site on an antigen to which B and/or T cells respond.
  • B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding 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.
  • Modification-Assisted Profiling also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (US 2004/0101920). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical mAbs, such that characterization can be focused on genetically distinct mAbs. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the anti-ANGPTL3 mAbs of the disclosure into groups of mAbs binding different epitopes.
  • ANGPTL3 contains an amino-terminal coiled-coil domain and a carboxyl-terminal fibrinogen like domain (FD) and the ANGPTL3 protein forms an oligomer in the absence of intermolecular disulfide bonds (Ge, et al., 2005, J Lipid Res, 46:1484-1490). It has been reported that the N-terminal coiled-coil domain is important in the inhibition of LPL activity (Ono, et al., 2003, J Biol Chem, 278:41804-41809).
  • the anti-hANGPTL3 antibody or antigen-binding fragment of an antibody binds an epitope within the N-terminal coiled-coil domain (residues 17-209) of hANGPTL3 (SEQ ID NO: 161) and neutralizes at least one activity of hANGPTL3 (e.g., inhibition of LPL activity).
  • the anti-hANGPTL3 antibody or antigen-binding fragment thereof binds an epitope within the N-terminal coiled-coil domain of hANGPTL3 and neutralizes at least one activity of hANGPTL3, with the proviso that the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO:170.
  • the antibody or fragment thereof specifically binds an epitope within residues 17 to 200, 17 to 100, 17 to 70, 17 to 65, 17 to 60, 17 to 57, 17 to 55, 17 to 50, 17 to 45, 17 to 40, or 17 to 35, of hANGPTL3 (SEQ ID NO:161), optionally with the proviso that the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO: 170.
  • the antibody or fragment thereof specifically binds an epitope within residues 40 to 200, 40 to 100, 40 to 70, 50 to 200, 50 to 100, 50 to 70, 58 to 200, 58 to 100, 58 to 70, 58 to 68, or 61 to 66 (known as a “heparin-binding motif”) of hANGPTL3 (SEQ ID NO:161), optionally with the proviso that the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO: 170.
  • the antibody or antibody fragment binds an epitope which may involve more than one of the enumerated epitopes or residues within the N-terminal coiled-coil region of hANGPTL3, optionally with the proviso that the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO: 170.
  • hANGPTL3 antibody or fragment thereof binds one or more fragments of hANGPTL3, for example, a fragment of at least 5 residues, at least 7 residues, at least 10 residues, at least 20 residues, at least 30 residues, at least 50 residues, at least 70 residues, at least 100 residues, at least 150 residues, or at least 200 residues, of hANGPTL3 (SEQ ID NO:161), optionally with the proviso that the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO: 170.
  • the present disclosure uses hANGPTL3 antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein. Likewise, the present disclosure also uses anti-hANGPTL3 antibodies that compete for binding to hANGPTL3 or a hANGPTL3 fragment with any of the specific exemplary antibodies described herein. [0082] One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference anti-hANGPTL3 antibody by using routine methods known in the art.
  • test antibody binds to the same epitope as a reference anti-hANGPTL3 antibody of the disclosure
  • the reference antibody is allowed to bind to a hANGPTL3 protein or peptide under saturating conditions.
  • the ability of a test antibody to bind to the hANGPTL3 molecule is assessed. If the test antibody is able to bind to hANGPTL3 following saturation binding with the reference anti- hANGPTL3 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-hANGPTL3 antibody.
  • test antibody may bind to the same epitope as the epitope bound by the reference anti-hANGPTL3 antibody used in the disclosure.
  • the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to a hANGPTL3 molecule under saturating conditions followed by assessment of binding of the test antibody to the hANGPTL3 molecule.
  • test antibody In a second orientation, the test antibody is allowed to bind to a hANGPTL3 molecule under saturating conditions followed by assessment of binding of the reference antibody to the ANGPTL3 molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the ANGPTL3 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to hANGPTL3.
  • an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.
  • Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans, et al., Cancer Res, 1990:50:1495-1502).
  • two antibodies have the same epitope if essentially 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.
  • Additional routine experimentation e.g., peptide mutation and binding analyses
  • peptide mutation and binding analyses can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding.
  • steric blocking or another phenomenon
  • this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.
  • the ANGPTL3 inhibitor is a human anti-ANGPTL3 monoclonal antibody conjugated to a therapeutic moiety (“immunoconjugate”), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope.
  • a therapeutic moiety such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope.
  • Cytotoxin agents include any agent that is detrimental to cells. Examples of suitable cytotoxin agents and chemotherapeutic agents for forming immunoconjugates are known in the art, see for example, WO 05/103081.
  • the ANGPTL3 inhibitors may be monospecific, bispecific, or multispecific antibodies.
  • Multispecific mAbs may be specific for different epitopes of one target polypeptide or may contain antigenbinding domains specific for more than one target polypeptide. See, e.g., Tutt, et al,. (1991) J. Immunol., 147:60-69.
  • the human anti-hANGPTL3 mAbs can be linked to or coexpressed with another functional molecule, e.g., another peptide or protein.
  • an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or a multispecific antibody with a second binding specificity.
  • An exemplary bi-specific antibody format that can be used in the context of the present disclosure involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference.
  • the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering).
  • the second C 3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second C H 3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of lgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of lgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of lgG4 antibodies. Variations on the bi- specific antibody format
  • the ANGPTL3 inhibitor is an anti-hANGPTL3 antibody or antigen-binding fragment thereof encompassing proteins having amino acid sequences that vary from those of the described mAbs, but that retain the ability to bind human ANGPTL3.
  • Such variant mAbs and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence but exhibit biological activity that is essentially equivalent to that of the described mAbs.
  • anti-hANGPTL3 antibodyencoding DNA sequences described herein encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, but that encode an anti-hANGPTL3 antibody or antibody fragment that is essentially bioequivalent to an anti-hANGPTL3 antibody or antibody fragment used in the disclosure. Examples of such variant amino acid and DNA sequences are discussed above.
  • Two antigen-binding proteins, or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied. In one embodiment, two antigenbinding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.
  • two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.
  • two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.
  • Bioequivalence may be demonstrated by in vivo and in vitro methods.
  • Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well- controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.
  • Bioequivalent variants of anti-hANGPTL3 antibodies used in the disclosure may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity.
  • cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation.
  • the disclosure provides therapeutic compositions comprising the anti-hANGPTL3 antibodies or antigen-binding fragments thereof of the present disclosure and the therapeutic methods using the same.
  • the administration of therapeutic compositions in accordance with the disclosure will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like.
  • suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like.
  • a multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.
  • vesicles such as LIPOFECTINTM
  • the dose may vary depending upon the age and the size of a subject to be administered, target disease, the purpose of the treatment, conditions, route of administration, and the like.
  • the antibody of the present disclosure is used for treating various conditions and diseases directly or indirectly associated with ANGPTL3, including severe hypertriglyceridemia, MCS, hypercholesterolemia, disorders associated with LDL and apolipoprotein B, and lipid metabolism disorders, and the like, in an adult patient, it is advantageous to intravenously or subcutaneously administer the antibody of the present disclosure at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight.
  • the frequency and the duration of the treatment can be adjusted.
  • the antibody or antigen-binding fragment thereof of the disclosure can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, to about 100 mg, or to about 50 mg.
  • the initial dose may be followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.
  • Various delivery systems are known and can be used to administer the pharmaceutical composition used in the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu, et al., (1987) J. Biol. Chem., 262:4429-4432).
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • Administration can be systemic or local.
  • the pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see Langer, (1990) Science, 249:1527-1533; Treat, et al., (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
  • a liposome see Langer, (1990) Science, 249:1527-1533; Treat, et al., (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra, Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201).
  • polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974).
  • a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).
  • the injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections.
  • aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
  • an alcohol e.g., ethanol
  • a polyalcohol e.g., propylene glycol, polyethylene glycol
  • a nonionic surfactant e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • oily medium there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • the injection thus prepared is preferably filled in an appropriate ampoule.
  • a pharmaceutical composition used in the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe.
  • a pen delivery device readily has applications in delivering a pharmaceutical composition used in the present disclosure.
  • Such a pen delivery device can be reusable or disposable.
  • a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition.
  • the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition.
  • the pen delivery device can then be reused.
  • a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
  • Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present disclosure. Examples include, but certainly are not limited to AUTOPENTM (Owen Mumford, Inc., Woodstock, UK), DISETRONICTM pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25TM pen, HUMALOGTM pen, HUMALIN 70/30TM pen (Eli Lilly and Co., Indianapolis, IN), NOVOPENTM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENTM, OPTIPEN PROTM, OPTIPEN STARLETTM, and OPTICLIKTM (Sanofi-Aventis, Frankfurt, Germany), to name only a few.
  • Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present disclosure include, but certainly are not limited to the SOLOSTARTM pen (Sanofi-Aventis), the FLEXPENTM (Novo Nordisk), and the KWIKPENTM (Eli Lilly).
  • the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients.
  • dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
  • the amount of the aforesaid antibody contained is generally about 0.1 to about 800 mg per dosage form in a unit dose; especially in the form of injection, the aforesaid antibody is contained in about 1 to about 500 mg, in about 5 to 300 mg, in about 8 to 200 mg, and in about 10 to about 100 mg for the other dosage forms.
  • the disclosure further provides therapeutic methods for treating or alleviating a symptom of severe hypertriglyceridemia in a subject, comprising administering an inhibitor of AngPTL3 to the subject.
  • severe hypertriglyceridemia is defined as a triglyceride (TG) concentration > 500 mg/dL (> 5.7 mmol/L) or a TG level >880 mg/dL (>10 mmol/L).
  • severe hypertriglyceridemia (sHTG) is defined as a triglyceride (TG) concentration > 1000 mg/dL.
  • LPL lipoprotein lipase
  • Familial chylomicronemia syndrome is a rare autosomal recessive genetic disorder resulting from two biallelic defects in LPL or LPL regulating genes, whereas multifactorial chylomicronemia syndrome (MCS) and sHTG are due to either one rare heterozygous large-effect variant or a high burden of common small-effect variants which predispose individuals to sHTG (Rosenson, et al. 2023 Pharmacol Therapeu 251 :108544).
  • VLDL very low-density lipoprotein
  • the general treatment regimen for sHTG includes dietary restrictions, lipid- lowering drug treatment (e.g., medium-chain triglycerides (MCT), fibrates, omega-3-fatty acids (omega-3-FA), and nicotinic acid).
  • MCT medium-chain triglycerides
  • fibrates do not offer fast onset of action, and fast-acting drugs like omega-3-FA and MCT may not be powerful enough to lower excessively elevated TG levels rapidly.
  • apheretic treatment (plasmapheresis) has also been considered (Ewald and Kloer 2012 Clin Res Cardiol Suppl 7(S1):31-35).
  • plasmapheresis is limited due to high cost and limited availability.
  • the disclosure further provides therapeutic methods for treating or alleviating a symptom of multifactorial chylomicronemia syndrome (MCS (also referred to as MFCS)) in a subject, comprising administering an inhibitor of AngPTL3 to the subject.
  • MCS multifactorial chylomicronemia syndrome
  • the symptom comprises elevated trigycerides.
  • Chylomicronemia syndrome is characterized by severe hypertriglyceridemia and fasting chylomicronemia (Goldberg and Chait 2020 Front Endocrinol (Lausanne) 11 :593931).
  • MCS multifactorial chylomicronemia syndrome
  • MCS is defined on the basis of a triglyceride (TG) concentration > 500 mg/dL (> 5.7 mmol/L) or a TG level >880 mg/dL (>10 mmol/L).
  • the disclosure also provides methods of reducing triglycerides in a subject with multifactorial chylomicronemia syndrome (MCS), comprising selecting a subject that exhibits elevated triglycerides and/or MCS, and administering an inhibitor of AngPTL3 to the subject.
  • MCS multifactorial chylomicronemia syndrome
  • the disclosure further provides therapeutic methods for treating diseases or disorders, which is directly or indirectly associated with hANGPTL3, by administering a hANGPTL3 antibody or fragment thereof of the disclosure in combination with one or more additional therapeutic agents.
  • the additional therapeutic agent may be one or more of any agent that is advantageously combined with one or more antibodies or fragments thereof of the disclosure, including HMG-CoA reductase inhibitors, such as cerovastatin, atorvastatin, simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin, pravastatin, and the like; niacin; various fibrates, such as fenofibrate, bezafibrate, ciprofibrate, clofibrate, gemfibrozil, and the like; LXR transcription factor activators, and the like.
  • hANGPTL3 antibody or fragment thereof used in the disclosure can be coadministered with other ANGPTL3 inhibitors as well as inhibitors of other molecules, such as ANGPTL4, ANGPTL5, ANGPTL6 and proprotein convertase subtilisin/kexin type 9 (PCSK9), which are involved in lipid metabolism, in particular, cholesterol and/or triglyceride homeostasis.
  • Inhibitors of these molecules include small molecules and antibodies that specifically bind to these molecules and block their activity (see, for example, anti-PCSK9 antibodies disclosed in U.S. 2010/0166768 A1).
  • the additional therapeutic agent may be one or more anti-cancer agents, such as chemotherapeutic agents, anti-angiogenic agents, growth inhibitory agents, cytotoxic agents, apoptotic agents, and other agents well known in the art to treat cancer or other proliferative diseases or disorders.
  • anti-cancer agents such as chemotherapeutic agents, anti-angiogenic agents, growth inhibitory agents, cytotoxic agents, apoptotic agents, and other agents well known in the art to treat cancer or other proliferative diseases or disorders.
  • anti-cancer agents include, but are not limited to, an anti-mitotic agent, such as docetaxel, paclitaxel, and the like; a platinum-based chemotherapeutic compound, such as cisplatin, carboplatin, iproplatin, oxaliplatin, and the like; or other conventional cytotoxic agent, such as 5- fluorouracil, capecitabine, irinotecan, leucovorin, gemcitabine, and the like, and anti- angiogenic agents, including vascular endothelial growth factor (VEGF) antagonists, such as anti-VEGF antibodies, e.g., bevacizumab (A VASTIN®, Genentech) and a receptorbased blocker of VEGF, e.g., “VEGF trap” described in US Patent No.
  • VEGF vascular endothelial growth factor
  • DII4 deltalike ligand 4
  • DII4 antagonists such as anti-DII4 antibodies as described in U.S. Patent Application Publication No. 2008/0181899
  • a fusion protein containing the extracellular domain of DII4 e.g., DII4-Fc as described in U.S. Patent Application Publication No.
  • inhibitors of receptor tyrosine kinases and/or angiogenesis including sorafenib (NEXAVAR® by Bayer Pharmaceuticals Corp.), sunitinib (SUTENT® by Pfizer), pazopanib (VOTRIENTTM by GlaxoSmithKline), toceranib (PALI_ADIA TM by Pfizer), vandetanib (ZACTIMATM by AstraZeneca), cediranib (RECENTIN® by AstraZeneca), regorafenib (BAY 73-4506 by Bayer), axitinib (AG013736 by Pfizer), lestaurtinib (CEP-701 by Cephalon), erlotinib (TARCEVA® by Genentech), gefitinib (IRESSATM by AstraZeneca), BIBW 2992 (TOVOKTM by Boehringer Ingelheim), lapatinib (TYKERB® by GlaxoSmith
  • hANGPTL3 antibody or fragment thereof of the disclosure may be also co-administered with the hANGPTL3 antibody or fragment thereof of the disclosure so as to ameliorate and/or reduce the symptoms accompanying the underlying cancer/tumor.
  • NSAIDS non-steroidal anti-inflammatory drugs
  • hANGPTL3 antibody or fragment thereof used in the disclosure and the additional therapeutic agent(s) can be co-administered together or separately. Where separate dosage formulations are used, the antibody or fragment thereof used in the disclosure and the additional agents can be administered concurrently, or separately at staggered times, i.e., sequentially, in appropriate orders.
  • VELOCIMMUNETM mice were immunized with human ANGPTL3, and the antibody immune response monitored by antigen-specific immunoassay using serum obtained from these mice.
  • Anti-hANGPTL3 expressing B cells were harvested from the spleens of immunized mice shown to have elevated anti-hANGPTL3 antibody titers and were fused with mouse myeloma cells to form hybridomas.
  • the hybridomas were screened and selected to identify cell lines expressing hANGPTL3-specific antibodies using assays as described below. The assays identified several cell lines that produced chimeric anti-hANGPTL3 antibodies, e.g., H1M896N.
  • Human ANGPTL3-specific antibodies were also isolated directly from antigen- immunized B cells without fusion to myeloma cells, as described in U.S. 2007/0280945 A1. Heavy and light chain variable regions were cloned to generate fully human anti- hANGPTL3 antibodies of lgG4 isotype designated as H4H1248P, H4H1250P, H4H1263S, H4H1268S, H4H1276S, H4H1279P, H4H1282P, H4H1292P, H4H1295P and H4H1296P. Stable recombinant antibody-expressing CHO cell lines were established.
  • HCVR Heavy Chain Variable Region
  • LCVR Light Chain Variable Region
  • Table 2 shows the heavy and light chain variable region amino acid sequence pairs of selected anti-hANGPTL3 antibodies and their corresponding antibody identifiers.
  • the N, P and S designations refer to antibodies having heavy and light chains with identical CDR sequences but with sequence variations in regions that fall outside of the CDR sequences (i.e., in the framework regions).
  • N, P and S variants of a particular antibody have identical CDR sequences within their heavy and light chain variable regions but contain modifications within the framework regions.
  • Cohort 1 comprised patients with familial chylomicronemia syndrome (FCS (with known bi-allelic LOF mutations in APOA5, APOC2, GPIHBP1, LMF1 , or LPL));
  • Cohort 2 comprised patients with MCS (with known heterozygous LOF mutations in APOA5, APOC2, GPIHBP1 , LMF1 , or LPL);
  • Cohort 3 comprised patients with MCS and without LPL pathway mutations. Initially, patients were enrolled into the afore-mentioned cohorts based on available genotype information from the patient’s medical history at screening. All patients were subsequently exome sequenced and analyzed.
  • the primary endpoint of the study was to determine the intra-patient percent change in mean serum triglycerides from baseline following 12 weeks of evinacumab treatment in Cohort 3 patients (the 12 weeks of treatment encompassed a combination of the DBTP and SBTP).
  • Cohort 3 was pre-specified as the analysis population for the primary endpoint analysis, as this group of patients had an intact LPL pathway and might respond optimally to evinacumab treatment.
  • baseline was defined as the geometric mean of all available triglyceride results at days -28, —14, and 1.
  • baseline was defined as the geometric mean of all available triglyceride results at weeks 6, 8, and 12.
  • percent change in other lipid/lipoprotein parameters from baseline to weeks 12 and 24 were also evaluated.
  • LDL-C was determined by ultracentrifugation; after separation of the very-low density lipoprotein (VLDL)/chylomicron subtraction by ultracentrifugation, LDL-C was determined as the cholesterol in the infranatant (performed by an enzymatic colorimetric assay) minus the HDL-C. ApoB and ApoA1 were assessed by nephelometry using a Siemens BNII nephelometer.
  • Genomic DNA was extracted from peripheral blood samples and submitted for whole exome sequencing. Briefly, 1pg of genomic DNA was fragmented and prepared for exome capture with a custom reagent kit from Kapa Biosystems. Samples were captured using the NimbleGen SeqCap VCRome 2.1 exome target design and sequenced using 75 bp paired-end sequencing on an Illumina HiSeq 2500 with v4 chemistry. Following sequencing, data was processed using a cloud-based pipeline developed at the RGC that uses DNAnexus and AWS to run standard tools for sample-level data production and analysis. Sequence reads were mapped and aligned to the GRCh37/hg19 human genome reference assembly using BWA-mem.
  • SNP and INDEL variants and genotypes were called using GATK’s HaplotypeCaller. Standard quality-control filters were applied to called variants. Passing variants were classified, annotated, and analyzed using an RGC implemented Mendelian analysis pipeline to evaluate their potential functional effects. Variants were annotated for their observed frequencies in population control databases such as dbSNP, the 1000 Genomes Project, the Exome Aggregation Consortium Database, and internal RGC databases in order to filter out common polymorphisms and high frequency, likely benign variants.
  • population control databases such as dbSNP, the 1000 Genomes Project, the Exome Aggregation Consortium Database, and internal RGC databases in order to filter out common polymorphisms and high frequency, likely benign variants.
  • Screened genes were: LPL, ANGPTL3, ANGPTL4, ANGPTL8, APOA1, APOA4, APOA5, APOC2, APOC3, APOD, COL18A1, CREB3L3, GALNT2, GPIHBP1, LMF1, PCSK7, MLXIPL, LIPI, USF1, ABCA 1, GPD1, GCKR, TRIB1, BTN2A 1, LRP8, TIMD4, ABCG5, ABCG8.
  • MRI was performed at baseline to assess pancreatic injury/inflammation through measurement of apparent diffusion coefficient (ADC) and levels of hepatic fat fraction.
  • ADC apparent diffusion coefficient
  • Patients underwent repeat MRI scans at 12 and 24 weeks of treatment to assess changes in pancreatic injury/inflammation and changes in liver hepatic fat fraction.
  • the primary endpoint was prespecified as the least squares mean percent reduction in triglycerides from baseline after 12 weeks of evinacumab exposure (combination of DBTP and SBTP) in Cohort 3. This was assessed in Cohort 3 using asynchronous study periods that were dependent upon treatment group in order to assess changes in triglycerides after 12 weeks of evinacumab treatment; for patients randomized to evinacumab, the primary analysis included the 12-week DBTP, while for patients randomized to placebo the primary analysis included the subsequent 12-week SBTP of active treatment with evinacumab. Point estimates of mean percent changes in triglycerides between the placebo run-in period and each observation were calculated using a mixed-effect model for repeated measures (MM RM) approach.
  • MM RM mixed-effect model for repeated measures
  • the primary endpoint analysis was based on the percent change in triglycerides from baseline following 12 weeks of treatment with evinacumab in Cohort 3. Point estimates of mean percent changes in triglycerides between the placebo run-in period and each observation were calculated using a mixed-effect model for repeated measures (MMRM) approach.
  • MMRM mixed-effect model for repeated measures
  • the MMRM model assessed within-patient treatment comparisons using an unstructured covariance matrix while accounting for baseline triglyceride values, study visit, and baseline triglyceride values by study visit interaction, but not trough levels of evinacumab, which varied and were often below the targeted threshold of 100 mg/L.
  • Post-hoc analyses were undertaken to evaluate whether (a) mean triglyceride values at baseline and week 12, (b) percent change in mean triglyceride values at week 12, (c) mean log-transformed triglyceride values from baseline and week 12, and (d) change in mean log-transformed triglyceride values from baseline at week 12 achieved normal distribution using the MMRM model. It was essential to determine if triglyceride values that do not follow a normal distribution are lognormal, as applying MMRM analysis without confirming the data obey a log-normal distribution, could lead to misinterpretation of data.
  • Coronary heart 1 (20.0) 1 (8.3) 2 (331) 1 (11.1) 1 (20.0) 5 (35.7) disease, n (%)
  • bln cludes omega-3-acid ethyl ester, omega-3 fatty acids, eicosapentaenoic acid ethyl ester, ezetimibe, fish oil, combination of docosahexaenoic acid, eicosapentaenoic acid and fish oil, eicosapentaenoic acid, and omega-3 triglycerides.
  • BMI body mass index
  • CHD coronary heart disease
  • IV intravenous
  • GLP-1 glucagon-like peptide- 1
  • Q4W every 4 weeks;
  • Asian 0 0 0 1 (11.1) 1 (20.0) 2 (16.7)
  • Coronary heart disease 1 (250) 1 (91) 2 (333) 1 (111) 1 (200) 4 (333) n (%)
  • Insulin 1 (25.0) 1 (9.1) 2(33.3) 4(44.4) 1 (20.0) 5 (41.7) (fast acting)
  • Insulin long 1 (25.0) 2(18.2) 3(50.0) 3(33.3) 3 (60.0) 4 (33.3) acting
  • GLP-1 inhibitors 0 (0.0) 0(0.0) 0(0.0) 1 (11.1) 1 (20.0) 1 (8.3) aTime from diagnosis to study randomization. includes omega-3-acid ethyl ester, omega-3 fatty acids, eicosapentaenoic acid ethyl ester, ezetimibe, fish oil, combination of docosahexaenoic acid, eicosapentaenoic acid and fish oil, eicosapentaenoic acid, and omega-3 triglycerides.
  • BMI body mass index
  • CHD coronary heart disease
  • DB double blind
  • GLP-1 glucagon-like peptide
  • P-value vs. 0.5045 0.8081 0.6114 placebo aPost-hoc nominal P-values are provided for descriptive purposes only.
  • bLDL-C was determined by ultracentrifugation.
  • Apo apolipoprotein
  • HDL-C high-density lipoprotein cholesterol
  • IV intravenous
  • LDL-C low-density lipoprotein cholesterol
  • Q1 first quartile
  • Q4W every 4 weeks
  • SD standard deviation
  • Intra-patient P-value ⁇ 0.0001 0.2834 0.3438
  • Intra-patient P-value 0 5781 1 0000 0 2783 aLDL-C was determined by ultracentrifugation.
  • Apo apolipoprotein
  • DB double blind
  • HDL-C high-density lipoprotein cholesterol
  • IV intravenous
  • LDL-C low-density lipoprotein cholesterol
  • Lp(a) lipoprotein(a)
  • Q1 first quartile
  • Q3 third quartile
  • Q4W every
  • Treatment exposure during the DBTP was generally consistent across the evinacumab and placebo groups.
  • the mean (SD) number of infusions were almost identical for the evinacumab and placebo groups (2.8 [0.6] infusions); the mean (SD) duration of study drug exposure was also almost identical between the evinacumab and placebo groups (11.4 [2.4] and 11.4 [2.3] weeks, respectively).
  • the pre-specified primary endpoint of this trial was the least squares mean percent reduction in triglycerides from baseline after 12 weeks of evinacumab exposure (combination of DBTP and SBTP) in Cohort 3.
  • the mean (standard error) percent reduction in triglycerides from baseline in Cohort 3 was -27.1% (37.4) (95% confidence interval [Cl]: -71.2 to 84.6).
  • the log transformed triglyceride values were not normally distributed, making use of mean percent change in triglyceride levels a less than ideal endpoint. Therefore, a post-hoc analysis was also performed using median percent reductions in triglyceride values.
  • Evinacumab was effective in reducing non-high-density lipoprotein cholesterol (non-HDL-C) in all three cohorts, including Cohort 1 , at week 12 (the end of the DBTP; Table 5; exploratory endpoint).
  • TEAEs treatment-emergent adverse events
  • Nasopharyngitis, AP, and type 2 diabetes mellitus occurred less frequently in the evinacumab than in the placebo group.
  • the treatment response to evinacumab was highly variable, in part influenced by the molecular etiology of sHTG.
  • Cohort 1 comprising patients with FCS due to biallelic mutations in known FCS genes
  • Cohort 3 no identified FCS gene mutations
  • the reduced response in Cohort 1 during the DBTP may be due to the markedly diminished LPL activity expected in these patients.
  • Patients in Cohort 3 observed to have a median reduction in triglycerides of -81.7% with evinacumab, are likely to have high polygenic risk scores for hypertriglyceridemia, whereas patients in Cohort 2 may have a combination of rare variants in the context of a high polygenic risk score background (Dron & Hegele 2020 Front Endocrinol (Lausanne) 11 :455; Dron, et al., 2019, J Clin Lipidol, 13:80-88).
  • the inter-individual variability in evinacumab exposure likely contributed to the observed variability in triglyceride-lowering response.
  • Patients with sHTG typically have increased ApoC3 levels due to elevation of triglyceride-rich lipoproteins carrying ApoC3.
  • evinacumab treatment in patients with sHTG substantially reduced plasma ApoC3 levels.
  • the ApoC3 reduction with evinacumab may have contributed to the triglyceride reduction in this population.
  • the reduction in ApoC3 observed was likely to be at least in part secondary to the reduction in TRLs brought about by evinacumab.
  • Treatment with evinacumab was generally well tolerated. During the DBTP, there were no clinically meaningful differences in TEAE frequency between the evinacumab and placebo groups. TEAEs also occurred in a similar proportion of patients in the DB evinacumab and DB placebo groups during the SBTP. Treatment with evinacumab had no effect on liver transaminases or hepatic fat as measured by MRI in contrast to a recent report of an antisense oligonucleotide targeted to ANGPTL3 (Bergmark, et al., 2022, Circulation, 145:1377-1386).

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Abstract

L'invention concerne l'utilisation d'inhibiteurs de l'ANGPTL3 pour réduire les triglycérides chez des sujets atteints du syndrome de chylomicronémie multifactorielle (MCS).
PCT/US2023/084357 2022-12-16 2023-12-15 Inhibiteurs de l'angptl3 pour la réduction des triglycérides dans le syndrome de chylomicronémie multifactorielle Ceased WO2024130165A1 (fr)

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