WO2024050526A1 - Compositions et méthodes de traitement du syndrome du qt long - Google Patents

Compositions et méthodes de traitement du syndrome du qt long Download PDF

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WO2024050526A1
WO2024050526A1 PCT/US2023/073337 US2023073337W WO2024050526A1 WO 2024050526 A1 WO2024050526 A1 WO 2024050526A1 US 2023073337 W US2023073337 W US 2023073337W WO 2024050526 A1 WO2024050526 A1 WO 2024050526A1
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seq
antibody
amino acid
acid sequence
set forth
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PCT/US2023/073337
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English (en)
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Jin Li
Mustafa KAMANI
Sylvia FONG
Bartlomiej BLUS
Choong-Ryoul SIHN
Tu Nguyen
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Biomarin Pharmaceutical Inc.
University Of Bern
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Publication of WO2024050526A1 publication Critical patent/WO2024050526A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • 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
    • C07K2317/72—Increased effector function due to an Fc-modification
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present application relates to materials and methods for the treatment of Long QT Syndrome.
  • LQTS Long QT syndrome
  • LQTS patients enter periods of electrical storms and are resistant to standard therapy and endure repeated defibrillation shocks and increased mortality (2). Additionally, patients with the type 2 form of LQTS respond less well to conventional treatment compared to LQTS1 individuals (10-15).
  • LQTS3 is caused by gain-of-function mutations in the SCN5A-encoded Navi.5 sodium ion (Na + ) channel.
  • Murine antibodies that bind to KCNQ1 are disclosed in PCT/IB2022/058286.
  • an isolated antibody that specifically binds KCNQ1 comprising a heavy chain variable region comprising an amino acid sequence that is at least 70% identical to an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence that is at least 70% identical to an amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 6 or SEQ ID NO: 9.
  • the antibody comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 6 or SEQ ID NO: 9.
  • the antibody comprises HCDR1 set forth in SEQ ID NO: 10, HCDR2 set forth in SEQ ID NO: 11, HCDR3 set forth in SEQ ID NO: 12; LCDR1 set forth in SEQ ID NO: 13, LCDR2 comprising the amino acid sequence “WAS,” and LCDR3 set forth in SEQ ID NO: 14.
  • the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5.
  • the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5.
  • the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8.
  • the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 6.
  • the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9.
  • the antibody comprises a heavy chain constant domain (e.g., IgGl, IgG2, IgG3 or IgG4).
  • the antibody comprises a light chain constant domain.
  • the heavy chain constant domain is a modified constant domain.
  • the disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody described herein and a pharmaceutically acceptable carrier, diluent or excipient.
  • nucleic acids encoding an antibody described herein, as well as vectors and host cells comprising vectors encoding the nucleic acids are also contemplated.
  • LQTS long QT syndrome
  • the long QT syndrome is LQTS1, LQTS2 or LQTS3.
  • the methods described herein optionally further comprise administering a standard of care to the subject for the treatment of long QT syndrome.
  • the standard of care is a beta-blocker, an implantable cardioverter-defibrillator (ICD), or a left cardiac sympathetic denervation.
  • the subject is also suffering from cardiomyopathy, diabetes, epilepsy or neurological comorbidities.
  • administering the antibody results in shorter cardiac repolarization compared to a subject that did not receive the antibody. In some embodiments, administering the antibody results in the reduced incidence of ventricular tachyarrhythmias including sudden cardiac arrest compared to a subject that did not receive the antibody. In some embodiments, administering the antibody does not affect KCNQ1 channel expression in the subject.
  • FIG. 1 Effects of KCNQ1 antibodies (30 pg/mL) on KCNQ1/KCNE1 current density. Representative current traces obtained in control conditions and in the presence of various antibody treatments. KCNQ1/KCNE1 transfected cells were incubated in the presence of KCNQ1 or IgG antibody for 24h prior to voltage clamp recordings. Antibody was kept in the external solution during the recordings. Same experimental conditions were applied for the control group. All currents were obtained using a 4 sec step protocol with pulses from -80 mV to +80 mV, followed by a repolarizing step to -40 mV for 2 sec. Holding potential was -90 mV and the interpulse interval was 15 sec. Dotted baselines denote the zero-current level.
  • FIG. 1 Effects of KCNQ1 antibodies (60 pg/mL) on KCNQ1/KCNE1 current density. Representative current traces obtained in control conditions and in the presence of various antibody treatments. KCNQ1/KCNE1 transfected cells were incubated in the presence of KCNQ1 or IgG antibody for 24h prior to voltage clamp recordings. Antibody was kept in the external solution during the recordings. Same experimental conditions were applied for the control group. All currents were obtained using a 4 sec step protocol with pulses from -80 mV to +80 mV, followed by a repolarizing step to -40 mV for 2 sec. Holding potential was -90 mV and the interpulse interval was 15 sec. Dotted baselines denote the zero-current level.
  • Figures 3A and 3B show the effects of KCNQ1 antibodies (30 pg/mL) on KCNQ1/KCNE1 current density and voltage dependence of activation. Data are shown as mean + SEM.
  • Figure 3 Current density as a function of voltage measured at the end of the 4-second depolarizing pulses in control (black) and various antibody treatments.
  • Figure 3B Conductance-voltage (G-V) relationships obtained from peak initial tail currents in control and various antibody treatments. G-V plots were fitted with a Boltzmann sigmoid equation to obtain the voltage at half-maximal activation (Vl/2) and slope (k).
  • Figures 4A and 4B show the effects of KCNQ1 antibodies (60 pg/mL) on KCNQ1/KCNE1 current density and voltage dependence of activation. Data are shown as mean + SEM.
  • Figure 4A Current density as a function of voltage measured at the end of the 4-second depolarizing pulses in control (black) and various antibody treatments.
  • Figure 4B Conductance-voltage (G-V) relationships obtained from peak initial tail currents in control and various antibody treatments. G-V plots were fitted with a Boltzmann sigmoid equation to obtain the voltage at half-maximal activation (Vl/2) and slope (k).
  • FIG. 1 Concentration-dependent effects of monoclonal antibodies on KCNQ1/KCNE1 step current density in HEK293 cells. Data shown are means +/- SEM. Statistical significance was tested with a two-way ANOVA (or mixed-effects model) followed by a Fisher’s Least Significant Difference (LSD) test.
  • FIG. 1 Concentration-dependent effects of monoclonal antibodies on KCNQ1/KCNE1 tail current density in HEK293 cells. Data are shown as mean +/- SEM. Tail current density obtained for each antibody treatment was measured as the initial peak current at -40 mV (normalized to cell capacitance) and plotted as a function of test potential. Statistical significance was tested with a two-way ANOVA (or mixed-effects model) followed by a Fisher’s Least Significant Difference (LSD) test.
  • LSD Least Significant Difference
  • Figures 7A and 7B shows the effect of KCNQ1 antibodies on KCNQ1/KCNE1 deactivation, at 30 pg/mL and 60 pg/mL, respectively. Data are shown as mean ⁇ SEM. The fraction of non-deactivated channels was estimated by dividing the current amplitude at the end of the tail current by the amplitude of the peak tail current. Greater ratios obtained in the presence of antibody compared to control reflect slower deactivation.
  • Figure 8 mAb-KCNQl peptide interactions were analyzed by 1 : 1 binding model and the reported KDs were derived from the antibody dissociation (k O ff) and association (k on ) rate constants. A table with mean KD values were calculated based on duplicate runs.
  • Figures 9A and 9B Binding kinetics and specificity of Ab-E mAh interactions with the KCNQ1 peptide, as monitored by Bio-Layer Interferometry. All measurements were performed in triplicates at 25°C in an assay-specific buffer using 96-well plates with an orbital shake speed of 1,000 rpm. To generate the binding curves, lOnM N-terminally biotinylated KCNQ1 peptide was immobilized on streptavidin- coated biosensor tips for 5 minutes, followed by a baseline equilibration for 5 minutes. The biosensor tips were then submerged in wells containing 0.2-14nM of antibody for 5 min to monitor the formation of the mAb-peptide complex.
  • Figure 9A The observed (or apparent) rate constant of Ab-E binding to the peptide is plotted as a linear function of antibody concentration (0.2-14nM), where the dashed lines represent the 95% confidence interval.
  • Figure 9B Specificity of the Ab-E-peptide interaction was determined using heat-denatured Ab-E as well as human IgGl and mouse IgG2a negative controls, all of which showed nonspecific background binding.
  • Figure 10 is a graph showing the results of a Clq binding immunoassay.
  • Figure 11 is a graph showing the results of a FcRn binding immunoassay.
  • Figure 12A is a graph showing the observed (or apparent) rate constant of Ab-G and Ab-F binding to the KCNQ1 peptide plotted as a linear function of antibody concentration.
  • Figures 12B ,12C, and 12D show the results of forced degradation analysis of Ab-F and Ab-G antibodies as compared to untreated samples.
  • Figure 12B summarizes the antibody forced degradation conditions
  • Figures 12C and 12D report the corresponding observed binding rates (kobs, s A (-l)) and the content of high molecular weight species (HMWS)by Size-Exclusion Ultrahigh Pressure Liquid Chromatography (SEC-UPLC).
  • Figure 13 is a graph showing the thermal melting profiles for Ab-E, Ab-F, and Ab-G antibodies, as monitored by differential scanning fluorimetry (DSF) using the UNCLE platform (Unchained Labs).
  • Figures 14A-14E depict representative current traces recorded from Ncytes cardiomyocytes in control conditions and following treatment with E-4031. Action potentials were recorded using the perforated patch technique at 1Hz, and at 35-37°C.
  • Figure 14A Action potentials recorded from a control cell (no antibody treatment) in the presence of E-4031 (25 nM). Action potentials are recorded in cells treated with 60 pg/mL muFl 1 ( Figure 14B) or 60 pg/mL hFl 1-5 ( Figure 14C) followed by E4031 (25 nM) challenge.
  • Figure 15 provides graphs showing the effects of muAb-1 and Ab-E) on Ncytes cardiomyocytes.
  • FIGs 16A-16E are graphs showing the effect of anti-KCNQl monoclonal antibodies on the baseline QT interval of rabbits.
  • Rabbits were treated either with Ab-E ( Figure 16A and Figure 16B), Ab-F ( Figure 16C), or Ab-G ( Figure 16D and Figure 16E), administered either (i) intravenously ( Figure 16A, Figure 16C, Figure 16D) or (ii) subcutaneously ( Figure 16B, Figure 16E).
  • ECG recordings were taken daily, and the change in QT interval from baseline (pre-dose) was plotted.
  • Figures 17A and 17B is a graph showing that Ab-E (2 mg/kg) protected against drug-induced QT prolongation and arrhythmia in rabbits.
  • Figure 18 are graphs showing the mean plasma concentration of antibodies Ab-E. Ab-F, and Ab- G over time.
  • the present disclosure is based on the discovery that anti-Potassium Voltage-Gated Channel Subfamily Q Member 1 (KCNQ1) monoclonal antibodies act as agonists on the IKS channel.
  • KCNQ1 anti-Potassium Voltage-Gated Channel Subfamily Q Member 1
  • the antibody may be any type of antibody, i.e., immunoglobulin, known in the art.
  • the antibody is an antibody of class or isotype IgA, IgD, IgE, IgG, or IgM.
  • the antibody described herein comprises one or more alpha, delta, epsilon, gamma, and/or mu heavy chains.
  • the antibody is an IgGl, IgG2, or IgG4 antibody.
  • the antibody described herein comprises one or more kappa or lambda light chains.
  • the term “specifically binds” as used herein means that the antibody (or antigen binding fragment) preferentially binds an antigen (e.g., KCNQ1) over other proteins. In some embodiments, “specifically binds” means the antibody has a higher affinity for the antigen than for other proteins.
  • Antibodies that specifically bind an antigen may have a binding affinity for the antigen of less than or equal to 1 x 10 7 M, less than or equal to 2 x 10 7 M, less than or equal to 3 x 10 7 M, less than or equal to 4 x 10 7 M, less than or equal to 5 x 10 7 M, less than or equal to 6 x 10 7 M, less than or equal to 7 x 10 7 M, less than or equal to 8 x 10 7 M, less than or equal to 9 x 10 7 M, less than or equal to 1 x 10 8 M, less than or equal to 2 x 10 8 M, less than or equal to 3 x 10 8 M, less than or equal to 4 x 10 8 M, less than or equal to 5 x 10 8 M, less than or equal to 6 x 10 8 M, less than or equal to 7 x 10 8 M, less than or equal to 8 x 10 8 M, less than or equal to 9 x 10 8 M, less than or equal to 1 x 10 9 M, less than or equal to
  • the antibody or antigen binding fragment thereof may bind KCNQ1 of SEQ ID NO: 1 with an affinity of about 1 x 10 7 M to about
  • the KCNQ1 is human KCNQ1 (Genbank Accession No. NP_000209, 676 amino acids, potassium voltage-gated channel subfamily KQT member 1 isoform 1).
  • a humanized antibody described herein comprises the following CDRs: HCDRlcomprising the amino acid sequence set forth in SEQ ID NO: 10; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12; LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13; LCDR2 comprising the amino acid sequence “WAS”; and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14.
  • the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 4.
  • the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 4. In some or any embodiments, the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 4. In some or any embodiments, the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 4 and retains the CDR amino acid sequences set out in SEQ ID NOs: 10-12.
  • the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 7. In some or any embodiments, the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 7. In some or any embodiments, the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 7.
  • the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 7 and retains the CDR amino acid sequences set out in SEQ ID NOs: 10-12.
  • the heavy chain variable region comprises one or more amino substitutions at one or more of residues 6, 32, 56, 60, and 92 of SEQ ID NO: 4 or SEQ ID NO: 7.
  • the humanized antibody comprises a light chain variable region comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some or any embodiments, the humanized antibody comprises a light chain variable region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 5. In some or any embodiments, the humanized antibody comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 5.
  • the humanized antibody comprises a light chain variable region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 5 and retains the CDR amino acid sequences set out in SEQ ID NOs: 13 and 14 and the LCDR2 amino acid sequence “WAS”.
  • the humanized antibody comprises a light chain variable region comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 6. In some or any embodiments, the humanized antibody comprises a light chain variable region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 6. In some or any embodiments, the humanized antibody comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 6.
  • the humanized antibody comprises a light chain variable region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 6 and retains the CDR amino acid sequences set out in SEQ ID NOs: 13 and 14 and the LCDR2 amino acid sequence “WAS”.
  • the humanized antibody comprises a light chain variable region comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 8. In some or any embodiments, the humanized antibody comprises a light chain variable region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 8. In some or any embodiments, the humanized antibody comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 8.
  • the humanized antibody comprises a light chain variable region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 8 and retains the CDR amino acid sequences set out in SEQ ID NOs: 13 and 14 and the LCDR2 amino acid sequence “WAS”.
  • the humanized antibody comprises a light chain variable region comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some or any embodiments, the humanized antibody comprises a light chain variable region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 9. In some or any embodiments, the humanized antibody comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 9.
  • the humanized antibody comprises a light chain variable region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 9 and retains the CDR amino acid sequences set out in SEQ ID NOs: 13 and 14 and the LCDR2 amino acid sequence “WAS”.
  • the light chain variable region comprises one or more amino substitutions at one or more of residues 31, 40, 57, 89, and 93, of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 9.
  • the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 4 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 5 (Antibody Ab-A).
  • the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 5 (Antibody Ab-B).
  • the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 8 (Antibody Ab-C).
  • the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 6 (Antibody Ab-D).
  • the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 9 (Antibody Ab-E).
  • the humanized antibody comprises a heavy chain constant region comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 17. In some or any embodiments, the humanized antibody comprises a heavy chain constant region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the humanized antibody comprises a heavy chain constant region comprising an amino acid sequence set forth in SEQ ID NO: 17.
  • the humanized antibody comprises a heavy chain constant region comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 21. In some or any embodiments, the humanized antibody comprises a heavy chain constant region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the humanized antibody comprises a heavy chain constant region comprising an amino acid sequence set forth in SEQ ID NO: 21.
  • the humanized antibody comprises a light chain constant region comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 19. In some or any embodiments, the humanized antibody comprises a light chain constant region comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain constant region comprising an amino acid sequence set forth in SEQ ID NO: 19.
  • the humanized antibody comprises a heavy chain comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 15. In some or any embodiments, the humanized antibody comprises a heavy chain comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the humanized antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 15.
  • the humanized antibody comprises a light chain comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 16. In some or any embodiments, the humanized antibody comprises a light chain comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the humanized antibody comprises a light chain comprising an amino acid sequence set forth in SEQ ID NO: 16.
  • the humanized antibody comprises a heavy chain comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some or any embodiments, the humanized antibody comprises a heavy chain comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the humanized antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 18.
  • the humanized antibody comprises a light chain comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some or any embodiments, the humanized antibody comprises a light chain comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the humanized antibody comprises a light chain comprising an amino acid sequence set forth in SEQ ID NO: 20.
  • the humanized antibody comprises a heavy chain comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some or any embodiments, the humanized antibody comprises a heavy chain comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the humanized antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 22.
  • the humanized antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 15 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 16.
  • the humanized antibody comprises the humanized antibody comprises a heavy chain variable domain set forth in SEQ ID NO: 4 or SEQ ID NO: 7 and a heavy chain constant region set forth in SEQ ID NO: 17.
  • the humanized antibody comprises a light chain variable region set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9 and a light chain constant region set forth in SEQ ID NO: 19.
  • the humanized antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 18 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 20.
  • the humanized antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 22 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 20.
  • the humanized antibody comprises a scFv (Vh-Vl) linkage set forth in SEQ ID NO: 23. In some embodiments, the humanized antibody comprises a scFv (VI- Vh) linkage set forth in SEQ ID NO: 24. In some embodiments, the humanized antibody comprising a Fab set forth in SEQ ID NO: 25.
  • Antigen binding fragments of the anti-KCNQl antibodies described herein are also contemplated.
  • the antigen binding fragment can be any part of an antibody that has at least one antigen binding site, and the antigen binding fragment may be part of a larger structure (an “antibody product”) that retains the ability of the antigen binding fragment to recognize KCNQ1.
  • antibody product an antibody product
  • these antibody products that include antigen binding fragments are included in the disclosure herein of “antigen binding fragment.”
  • Examples of antigen binding fragments include, but are not limited to, Fab, F(ab')2, a monospecific or bispecific Falv. a trispecific Fab ;.
  • the antigen binding fragment is a domain antibody, VhH domain, V-NAR domain, VH domain, VL domain, or the like.
  • Antibody fragments of the disclosure are not limited to these exemplary types of antibody fragments.
  • antigen binding fragment is a Fab fragment.
  • the antigen binding fragment comprises two Fab fragments. In exemplary aspects, the antigen binding fragment comprises two Fab fragments connected via a linker. In exemplary aspects, the antigen binding fragment comprises or is a minibody comprising two Fab fragments. In exemplary aspects, the antigen binding fragment comprises, or is, a minibody comprising two Fab fragments joined via a linker. Minibodies are known in the art. See, e.g., Hu et al., Cancer Res 56: 3055-3061 (1996). In exemplary aspects, the antigen binding fragment comprises or is a minibody comprising two Fab fragments joined via a linker, optionally, comprising an alkaline phosphatase domain.
  • a domain antibody comprises a functional binding unit of an antibody, and can correspond to the variable regions of either the heavy (VH) or light (VL) chains of antibodies.
  • a domain antibody can have a molecular weight of approximately 13 kDa, or approximately one-tenth of a full antibody. Domain antibodies may be derived from full antibodies such as those described herein.
  • Suitable methods of making antibodies are known in the art. For instance, standard hybridoma methods are described in, e.g., Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). Monoclonal antibodies for use in the methods of the disclosure may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (Proc Natl Acad Sci 86: 3833-3837; 1989), and Winter G and Milstein C (Nature 349: 293- 299, 1991). If the full sequence of the antibody or antigen-binding fragment is known, then methods of producing recombinant proteins may be employed. See, e.g., “Protein production and purification” Nat Methods 5(2): 135-146 (2008).
  • the antibodies (or antigen binding fragments) are isolated from cell culture or a biological sample if generated in vivo.
  • Phage display also can be used to generate the antibody of the present disclosures.
  • phage libraries encoding antigen-binding variable (V) domains of antibodies can be generated using standard molecular biology and recombinant DNA techniques (see, e.g., Sambrook et al. (eds.), Molecular Cloning, A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, New York (2001)).
  • Phage encoding a variable region with the desired specificity are selected for specific binding to the desired antigen, and a complete or partial antibody is reconstituted comprising the selected variable domain.
  • Nucleic acid sequences encoding the reconstituted antibody are introduced into a suitable cell line, such as a myeloma cell used for hybridoma production, such that antibodies having the characteristics of monoclonal antibodies are secreted by the cell (see, e.g., Janeway et al., supra, Huse et al., supra, and U.S. Patent 6,265,150).
  • a suitable cell line such as a myeloma cell used for hybridoma production
  • Related methods also are described in U.S. Patent No. 5,403,484; U.S. Patent No. 5,571,698; U.S. Patent No. 5,837,500; U.S. Patent No. 5,702,892.
  • Antibodies can be produced by transgenic mice that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods are known in the art and described in, for example U.S. Patent Nos. 5,545,806 and 5,569,825, and Janeway et al., supra.
  • Chemically constructed bispecific antibodies may be prepared by chemically cross-linking heterologous Fab or F(ab')2 fragments by means of chemicals such as heterobifunctional reagent succinimidyl-3-(2-pyridyldithiol)-propionate (SPDP, Pierce Chemicals, Rockford, Ill.).
  • the Fab and F(ab')2 fragments can be obtained from intact antibody by digesting it with papain or pepsin, respectively (Karpovsky et al., J. Exp. Med. 160:1686-701 (1984); Titus et al., J. Immunol., 138:4018-22 (1987)).
  • Methods of testing antibodies for the ability to bind to an epitope of KCNQ1 are known in the art and include, e.g., radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, surface plasmon resonance (e.g., Biacore), and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002/0197266).
  • RIA radioimmunoassay
  • ELISA ELISA
  • Western blot Western blot
  • immunoprecipitation e.g., surface plasmon resonance
  • Biacore surface plasmon resonance
  • competitive inhibition assays see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002/0197266).
  • Antibody fragments that contain the antigen binding, or idiotype, of the antibody molecule may be generated by techniques known in the art. For example, a F(ab')2 fragment may be produced by pepsin digestion of the antibody molecule; Fab' fragments may be generated by reducing the disulfide bridges of the F(ab')2 fragment; and two Fab' fragments which may be generated by treating the antibody molecule with papain and a reducing agent.
  • the disclosure is not limited to enzymatic methods of generating antigen binding fragments; the antigen binding fragment may be a recombinant antigen binding fragment produced by expressing a polynucleotide encoding the fragment in a suitable host cell.
  • a single-chain variable region fragments which consists of a truncated Fab fragment comprising the variable (V) domain of an antibody heavy chain linked to a V domain of an antibody light chain via a synthetic peptide, can be generated using routine recombinant DNA technology techniques (see, e.g., Janeway et al., supra).
  • dsFv disulfide-stabilized variable region fragments
  • dsFv can be prepared by recombinant DNA technology (see, e.g., Reiter et al., Protein Engineering, 7, 697-704 (1994)).
  • Recombinant antibody fragments e.g., scFvs
  • scFvs can also be engineered to assemble into stable multimeric oligomers of high binding avidity and specificity to different target antigens.
  • diabodies dimers
  • triabodies trimers
  • tetrabodies tetramers
  • the heavy chains of the humanized antibodies described herein may further comprise one or more mutations that affect binding of the antibody containing the heavy chains to one or more Fc receptors.
  • One of the functions of the Fc portion of an antibody is to communicate to the immune system when the antibody binds its target. This is commonly referenced as “effector function.” Communication leads to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement dependent cytotoxicity (CDC).
  • ADCC and ADCP are mediated through the binding of the Fc to Fc receptors on the surface of cells of the immune system.
  • CDC is mediated through the binding of the Fc with proteins of the complement system, e.g., Clq.
  • the effector function of an antibody can be increased, or decreased, by introducing one or more mutations into the Fc.
  • Embodiments of the invention include heterodimeric antibodies, having an Fc engineered to increase effector function (U.S. 7,317,091 and Strohl, Curr. Opin. Biotech., 20:685-691, 2009; both incorporated herein by reference in its entirety).
  • Fc molecules having increased effector function include those having a mutation (e.g., substitution) at one or more of the following residues [numbering based on the EU numbering scheme]: 228, 234, 235, 236, 237, 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 282, 283, 285, 298, 289, 290,
  • Fc molecules having increased effector function include those having one or more of the following substitutions [numbering based on the EU numbering scheme for an IgGl]:
  • K326W/E333S [0100] K290E/S298G/T299A
  • the humanized antibodies have an Fc engineered to decrease effector function.
  • Exemplary Fc molecules having decreased effector function include those having one or more of the following substitutions [numbering based on the EU numbering scheme]:
  • V234A/G237A (IgG2)
  • Another method of increasing effector function of IgG Fc-containing proteins is by reducing the fucosylation of the Fc. Removal of the core fucose from the biantennary complex-type oligosachharides attached to the Fc greatly increased ADCC effector function without altering antigen binding or CDC effector function.
  • Several methods are known for reducing or abolishing fucosylation of Fc-containing molecules, e.g., antibodies.
  • a composition comprises an antibody having reduced fucosylation or lacking fucosylation altogether.
  • the humanized antibodies have an Fc having one or more of the following substitutions (IgGl): N297D N297G N297A N297Q
  • Suitable methods of making antibodies are known in the art. For instance, standard hybridoma methods are described in, e.g., Harlow and Fane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). Monoclonal antibodies for use in the methods of the disclosure may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (Proc Natl Acad Sci 86: 3833-3837; 1989), and Winter G and Milstein C (Nature 349: 293- 299, 1991). If the full sequence of the antibody or antigen-binding fragment is known, then methods of producing recombinant proteins may be employed. See, e.g., “Protein production and purification” Nat Methods 5(2): 135-146 (2008). In some embodiments, the antibodies (or antigen binding fragments) are isolated from cell culture or a biological sample if generated in vivo.
  • Phage display also can be used to generate the antibody of the present disclosures.
  • phage libraries encoding antigen-binding variable (V) domains of antibodies can be generated using standard molecular biology and recombinant DNA techniques (see, e.g., Sambrook et al. (eds.), Molecular Cloning, A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, New York (2001)).
  • Phage encoding a variable region with the desired specificity are selected for specific binding to the desired antigen, and a complete or partial antibody is reconstituted comprising the selected variable domain.
  • Nucleic acid sequences encoding the reconstituted antibody are introduced into a suitable cell line, such as a myeloma cell used for hybridoma production, such that antibodies having the characteristics of monoclonal antibodies are secreted by the cell (see, e.g., Janeway et al., supra, Huse et al., supra, and U.S. Patent 6,265,150).
  • a suitable cell line such as a myeloma cell used for hybridoma production
  • Related methods also are described in U.S. Patent No. 5,403,484; U.S. Patent No. 5,571,698; U.S. Patent No. 5,837,500; U.S. Patent No. 5,702,892.
  • Patent No. 5,858,657 U.S. Patent No. 5,871,907; U.S. Patent No. 5,969,108; U.S. Patent No. 6,057,098; and U.S. Patent No. 6,225,447.
  • Antibodies can be produced by transgenic mice that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods are known in the art and described in, for example U.S. Patent Nos. 5,545,806 and 5,569,825, and Janeway et al., supra.
  • Antibodies described herein may be monovalent or multivalent and can be made using the knob-in hole method.
  • Typical knob in hole antibodies are made by altering residues in the CH3 region of the Fc to allow for better binding between heavy chain residues of a heterodimeric antibody, one containing a “knob” and the other containing a “hole”. See e.g., Elliott et al., J. Mol Biol 426: 1947-1957, 2014).
  • knob in hole changes can refer to a T366W (“knob”) change, as well as T366S, L368A, and Y407V (“hole”) alterations.
  • S354C and Y349C cyste replacement mutations at CH3 region of “knob” and “hole”, respectively.
  • An exemplary antibody chain with knob in hole modification is set out in SEQ ID NO: SEQ ID NO: 27 and SEQ ID NO: 28.
  • Sequences encoding particular antibodies can be used for transformation of a suitable mammalian host cell, such as a CHO cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference). The transformation procedure used depends upon the host to be transformed.
  • Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, Sp2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines.
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • Sp2/0 cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • Hep G2 human hepatocellular carcinoma cells
  • Cell lines of particular preference are selected through determining which cell lines have high expression levels and produce antibodies with KCNQ1 -binding properties.
  • the disclosure provides a nucleic acid comprising a nucleotide sequence that encodes the heavy chain variable region and/ or light chain variable region of a humanized antibody as described herein.
  • nucleic acid encoding the antibody comprising the nucleic acid encoding the antibody.
  • nucleic acid encoding a heavy chain variable region and light chain variable region are expressed on the same vector or different vectors.
  • a host cell comprising a nucleic acid encoding a humanized antibody heavy and/or light chain variable region or vector expressing said nucleic acid.
  • the host cell is an eukaryotic cell.
  • the disclosure provides a method of using the humanized antibody or fragment thereof described herein to measure the amount of KCNQ1 in a sample.
  • a biological sample from a mammalian subject is contacted with a humanized anti-KCNQl antibody (or antigen binding fragment thereof) described herein for a time sufficient to allow immunocomplexes to form. Immunocomplexes formed between the humanized antibody and KCNQ1 in the sample are then detected.
  • the amount of KCNQ1 in the biological sample is optionally quantitated by measuring the amount of the immunocomplex formed between the human antibody and the KCNQ1.
  • the humanized antibody can be quantitatively measured if it has a detectable label, or a secondary antibody can be used to quantify the immunocomplex.
  • the biological sample comprises a tissue sample, a cell sample, or a biological fluid sample, such as blood, saliva, serum, or plasma.
  • Conditions for incubating an antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody used in the assay.
  • One skilled in the art will recognize that any one of the commonly available immunological assay formats can readily be adapted to employ the antibodies (or fragments thereof) of the present disclosure. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G.R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol.
  • test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or fluids used as the sample to be assayed.
  • the assay described herein may be useful in, e.g., evaluating the efficacy of a particular therapeutic treatment regime in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • the humanized KCNQ1 antibody (or antigen binding fragment thereof) is attached to a solid support, and binding is detected by detecting a complex between the KCNQ1 and the humanized antibody (or antigen binding fragment thereof) on the solid support.
  • the humanized antibody (or fragment thereof) optionally comprises a detectable label and binding is detected by detecting the label in the KCNQ1 -antibody complex.
  • Detection of the presence or absence of a KCNQl-antibody complex can be achieved by using any method known in the art.
  • the transcript resulting from a reporter gene transcription assay of a KCNQ1 peptide interacting with a target molecule typically encodes a directly or indirectly detectable product (e.g., P-galactosidase activity and luciferase activity).
  • a target molecule e.g., antibody
  • a directly or indirectly detectable product e.g., P-galactosidase activity and luciferase activity.
  • one of the components usually includes, or is coupled to, a detectable label.
  • labels can be used, such as those that provide direct detection (such as radioactivity, luminescence, optical or electron density) or indirect detection (such as epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase).
  • the label can be bound to the antibody, or incorporated into the structure of the antibody.
  • label can be detected while bound to the solid substrate or subsequent to separation from the solid substrate.
  • Labels can be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers or indirectly detected with antibody conjugates, or streptavidin-biotin conjugates. Methods for detecting the labels are well known in the art.
  • compositions comprising a humanized KCNQ1 antibody or antigen binding fragment thereof described herein are also contemplated.
  • the pharmaceutical composition contains formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, proline, methionine or lysine); antimicrobials; antioxidants (such as reducing agents, oxygen/free-radical scavengers, and chelating agents (e.g., ascorbic acid, EDTA, sodium sulfite or sodium hydrogen-sulfite)); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, man
  • Selection of the particular formulation materials described herein may be driven by, for example, the intended route of administration, delivery format and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra.
  • the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and may further include sorbitol or a suitable substitute therefor.
  • the composition may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake or an aqueous solution.
  • optional formulation agents REMINGTON'S PHARMACEUTICAL SCIENCES, supra
  • the antibody or (antigen binding fragment thereof) may be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • compositions of the invention can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the digestive tract, such as orally. Preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • the formulation components are present preferably in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • the composition may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired antibody or fragment in a pharmaceutically acceptable vehicle.
  • a particularly suitable vehicle for parenteral injection is sterile distilled water in which the antibody or fragment is formulated as a sterile, isotonic solution, properly preserved.
  • implantable drug delivery devices may be used to introduce the desired antibody (or antigen binding fragment thereof).
  • compositions including formulations involving antigen binding proteins in sustained- or controlled-delivery formulations are contemplated herein.
  • Techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, bio- erodible microparticles or porous beads and depot injections, are available in the art. See, for example, International Patent Application No. PCT/US93/00829, which is incorporated by reference and describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions.
  • Sustained-release preparations may include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • Sustained release matrices may include polyesters, hydrogels, polylactides (as disclosed in U.S. Pat. No. 3773919 and European Patent Application Publication No. EP058481, each of which is incorporated by reference), copolymers of L-glutamic acid and gamma ethyl- L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech.
  • Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European Patent Application Publication Nos. EP036676; EP088046 and EP143949, incorporated by reference.
  • Embodiments of the humanized antibody formulations can further comprise one or more preservatives.
  • compositions described herein will be via any common route so long as the target tissue is available via that route.
  • the pharmaceutical compositions may be introduced into the subject by any conventional method, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., term release); by subcutaneous, oral, sublingual, nasal, anal, vaginal, or transdermal delivery, or by surgical implantation at a particular site.
  • one or more doses of the humanized antibody or antigen binding fragment are administered in an amount and for a time effective to treat a long QT syndrome (LQTS) in a subject.
  • LQTS long QT syndrome
  • one or more administrations of an antibody or antigen binding fragment thereof described herein are optionally carried out over a therapeutic period of, for example, about 1 week to about 24 months (e.g., about 1 month to about 12 months, about 1 month to about 18 months, about 1 month to about 9 months or about 1 month to about 6 months or about 1 month to about 3 months).
  • a subject is administered one or more doses of an antibody or fragment thereof described herein over a therapeutic period of, for example about 1 month to about 12 months (52 weeks) (e.g., about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, or about 11 months).
  • the antibody or fragment thereof is administered periodically over a time period of one year (12 months, 52 weeks) or less (e.g., 9 months or less, 6 months or less, or 3 months or less).
  • the antibody or fragment thereof is administered to the human once every about 3 days, or about 7 days, or 2 weeks, or 3 weeks, or 4 weeks, or 5 weeks, or 6 weeks, or 7 weeks, or 8 weeks, or 9 weeks, or 10 weeks, or 11 weeks, or 12 weeks, or 13 weeks, or 14 weeks, or 15 weeks, or 16 weeks, or 17 weeks, or 18 weeks, or 19 weeks, or 20 weeks, or 21 weeks, or 22 weeks, or 23 weeks, or 6 months, or 12 months.
  • one or more doses comprising from about 50 milligrams to about 1,000 milligrams of the antibody or antigen binding fragment thereof are administered to a subject (e.g., a human subject).
  • a dose can comprise at least about 5 mg, at least about 15 mg, at least about 25 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 120 mg, at least about 150 mg, at least about 200 mg, at least about 210 mg, at least about 240 mg, at least about 250 mg, at least about 280 mg, at least about 300 mg, at least about 350 mg, at least about 400 mg, at least about 420 mg, at least about 450 mg, at least about 500 mg, at least about 550 mg, at least about 600 mg, at least about 650 mg, at least about 700 mg, at least about 750 mg, at least about 800 mg, at least about 850 mg, at least about 900 mg, at
  • Ranges between any and all of these endpoints are also contemplated, e.g., about 50 mg to about 80 mg, about 70 mg to about 140 mg, about 70 mg to about 270 mg, about 75 mg to about 100 mg, about 100 mg to about 150 mg, about 140 mg to about 210 mg, or about 150 mg to about 200 mg, or about 180 mg to about 270 mg.
  • the dose is administered at any interval, such as multiple times a week (e.g., twice or three times per week), once a week, once every two weeks, once every three weeks, or once every four weeks.
  • the one or more doses can comprise between about 0.1 to about 50 milligrams (e.g., between about 5 and about 50 milligrams), or about 1 to about 100 milligrams, of antibody (or antigen binding fragment thereof) per kilogram of subject body weight (mg/kg).
  • the dose may comprise at least about 0.1 mg/kg, at least about 0.5 mg/kg, at least about 1 mg/kg, at least about 2 mg/kg, at least about 3 mg/kg, at least about 4 mg/kg, at least about 5 mg/kg, at least about 6 mg/kg, at least about 7 mg/kg, at least about 8 mg/kg, at least about 9 mg/kg, at least about 10 mg/kg, at least about 11 mg/kg, at least 12 mg/kg, at least 13 mg/kg, at least 14 mg/kg, at least about 15 mg/kg, at least 16 mg/kg, at least 17 mg/kg, at least 18 mg/kg, at least 19 mg/kg, at least about 20 mg/kg, at least 21 mg/kg, at least 22 mg/kg, at least 23 mg/kg, at least 24 mg/kg, at least about 25 mg/kg, at least about 26 mg/kg, at least about 27 mg/kg, at least about 28 mg/kg, at least about 29 mg/kg, at least about 30 mg/kg, at least about
  • Ranges between any and all of these endpoints are also contemplated, e.g., about 1 mg/kg to about 3 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 8 mg/kg, about 3 mg/kg to about 8 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kg to about 40 mg/kg, about 5 mg/kg to about 30 mg/kg, or about 5 mg/kg to about 20 mg/kg.
  • LQTS long QT syndrome
  • the long QT syndrome is LQTS2 or LQTS3. In some embodiments, the long QT syndrome is LQTS2. In some embodiments, the long QT syndrome is LQTS3.
  • the subject is also suffering from cardiomyopathy, diabetes, epilepsy or neurological comorbidities.
  • administering the antibody results in shorter cardiac repolarization compared to a subject that did not receive the antibody.
  • administering the antibody results in the reduced incidence of ventricular tachyarrhythmias including sudden cardiac arrest compared to a subject that did not receive the antibody.
  • the subject is suffering from LQT3. In some embodiments, the subject has acquired long QT. In some embodiments, the subject has acute QT prolongation.
  • administering the humanized antibody results in shorter cardiac repolarization (QT or JT interval on ECG, or variations thereof such as QT interval corrected by Bazett formula (QT/RR 172 ), Fridericia (QT/RR 173 ), Framingham (QT+0.154(l-RR)), Hodges (QT+1.75(HR-60), Rautahaiju (QTx(120+HR)/180), heart rate-corrected JT (QTc-QRS)) of the subject by at least 5% (or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50% or more) compared to the cardiac repolarization of the subject at baseline.
  • administering the humanized antibody does not affect KCNQ1 channel expression in the subject.
  • the antibody has no detectable or minimal off-target effects, e.g. epilepsy, neuropsychiatric comorbidities, diabetes mellitus or impaired glucose tolerance, thyroid disorder.
  • the humanized antibodies disclosed here can be administered alone or optionally in combination with other therapeutic agents useful for the treatment of LQTS.
  • any active agent known to be useful in treating a condition, disease, or disorder described herein can be used in the methods of the invention, and either combined with the amino sterol compositions used in the methods of the invention, or administered separately or sequentially.
  • Exemplary additional agents include, but are not limited to, betablockers such as propanolol (Inderal®), atenolol (Tenormin®), metoprolol (Metoprolol®, Lopressor®), nadolol (Corgard®), bisoprolol (Zebeta®, Monocor®); antiarrhythmics such mexiletine (Mexitil®), ranolazine (Ranexa®); calcium channel blockers such as diltiazem (Cardizem®) and verapamil (Verelan®); and digitalis derived drugs such as digoxin (Lanoxin®).
  • betablockers such as propanolol (Inderal®), atenolol (Tenormin®), metoprolol (Metoprolol®, Lopressor®), nadolol (Corgard®), bisoprolol (Zebeta®, Monocor®); antiarrhythmics such mexiletine
  • the antibody is administered in combination with an agent that would otherwise prolong the QT interval of patients.
  • kits for producing a single-dose administration unit may each contain both a first container having a dried protein and a second container having an aqueous formulation.
  • kits containing single and multi-chambered pre-filled syringes e.g., liquid syringes and lyosyringes are provided.
  • TsA201 cells transformed human embryonic kidney 293 cells
  • MEM modified Eagle’s medium
  • penicillin 100 ⁇ g/ml
  • streptomycin 100 ⁇ g/ml
  • amphotorecin 100 ⁇ g/ml amphotorecin.
  • TsA201 cells are HEK293 cells stably transfected with the SV40 large tumor antigen allowing higher level of expression of vectors containing the SV40 vector such as pCDNA3.1 used in our constructs. Cells were maintained at 37°C in an air/5% CO2 incubator.
  • Green Fluorescent Protein cDNA (GFP, 1 pg) was co-transfected along with KCNQ1 and KCNE1 to identify transfected cells. Antibody treatment was started 24h after transfection, for a 24h period.
  • Electrophysical procedures Coverslips containing cells were removed from the incubator and placed in a superfusion chamber (volume 250 pl) containing the external bath solution (See Table 2 for composition of the recording solution) maintained at room temperature. Test antibody (30 ug/mL) was present in the external solution throughout the experiment. Whole-cell current recordings were performed using an Axopatch 200B amplifier. Patch electrodes were pulled from thin- walled borosilicate glass on a horizontal micropipette puller and fire-polished. Electrodes had resistances of 1.5-3.0 mQ when filled with control filling solution (See Table 2 for composition of the internal solution). Analog capacity compensation and 60% - 85% series resistance compensation was used in all measurements. Data were sampled at 10-20 kHz and filtered at 5 to 10 kHz before digitization and stored on a computer for later analysis using pClamplO software.
  • I-V current-voltage
  • Antibodies The list of test antibodies is shown in Table 3. Antibody solutions were aliquoted before use to avoid multiple freeze/thaw cycles. Aliquots were stored at -80°C. Test antibodies were formulated in 100 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH6.0. Human IgG isotype control was purchased from ThermoFisher Scientific. Test antibodies were tested at 30 pg/mL.
  • V is the potential
  • V 1/2 is the voltage where channels exhibit half-maximal activation (between bottom and top)
  • k is a slope factor reflecting the voltage range over which an e-fold change in open probability (Po) is observed. Results are shown as mean ⁇ SEM unless specified otherwise. Current density (pA/pF) was obtained by dividing current amplitude by cell capacitance. Statistical analysis: A one way ANOVA with Dunnett’s multiple comparisons test was performed to determine statistical significance between activation V1/2 calculated in control and in each antibody treatment.
  • Binding kinetics of KCNQ1 peptide (Nterm-Biotin-(CH2O)4-AEKDAVNESGRVEFGSYADA- Cterm, SEQ ID NO: 2) interactions with seven monoclonal antibodies (mAbs), the parental muAb-1 and its humanized variants (Ab- A, Ab-B, Ab-C, Ab-D, Ab-E, Ab-F, and Ab-G), was monitored using Octet RED96 Bio-Layer Interferometry (Sartorius). All measurements were performed in duplicate at 25°C in an assay-specific buffer containing lx PBS pH 7.4, lx kinetic buffer and 1% BSA using 96-well plates with orbital shake speed of 1,000 rpm.
  • the binding curves were generated by first immobilizing ImM N- terminally biotinylated KCNQ1 peptide on streptavidin-coated biosensor tips for 2.5min followed by a baseline equilibration step for 5 min. The biosensor tips were then submerged in wells containing 100 nM mAbs for 5 min to monitor formation of the mAb-peptide complex, followed by antibody dissociation in the assay buffer for 5 minutes. A shift in the interference pattern of white light reflected from the surface of a biosensor tip caused by antibody binding/dissociation was monitored in real time.
  • Antibody-peptide interactions were analyzed by a 1 : 1 binding model and the dissociation constants (KDs) were determined as ratios of dissociation (koff) and association (kon) binding rate constants derived using non-linear fitting model in GraphPad Prism.
  • KDs dissociation constants
  • patch clamp recordings are performed at 35-37°C, between day 7 and 14 post-thawing, on spontaneously beating cells.
  • the amphotericin B -perforated patch method is used to record action potentials under current-clamp conditions with dPatch amplifier controlled by SutterPatch (Sutter instruments, Novato, USA).
  • hiPSC-CMC are maintained in external solution containing (mmol/E): 140 NaCl, 5 KC1, 1 MgCh. 10 HEPES, 1.8 CaCh, 10 glucose (pH 7.4 adjusted with NaOH) ⁇ humanized antibody (30 and 60 ⁇ g/ml, 24h).
  • Borosilicate glass pipettes (tip resistances of 2-4MQ) are filled with amphotericin B (200 ⁇ g/ml) containing internal solution.
  • the internal solution is composed of (mmol/L): 110 K+aspartate, 20 KCL, 1 MgCh, 5 Mg2+ATP, 0.1 Li+GTP, 10 HEPES, 5 Na+phosphocreatine, 0.05 EGTA (pH adjusted to 7.3 with KOH).
  • the cells are challenged with 25nmol/L E-4031 (Alomone Labs, Jerusalem, Israel). APD is determined at 90% (APD90) repolarization, excluding cells with AP alternans.
  • Example 4 Functional assessment of humanized anti-KCNQl antibodies in LQT3 patient-derived cells
  • patch clamp recordings are performed at 35-37°C, between day 7 and 14 post-thawing, on spontaneously beating cells.
  • the amphotericin B -perforated patch method is used to record action potentials under current-clamp conditions with dPatch amplifier controlled by SutterPatch (Sutter instruments, Novato, USA).
  • hiPSC-CMC are maintained in external solution containing (mmol/L): 140 NaCl, 5 KC1, 1 MgCL, 10 HEPES, 1.8 CaCh, 10 glucose (pH 7.4 adjusted with NaOH) ⁇ humanized antibody (30 and 60 ⁇ g/ml, 24h).
  • Borosilicate glass pipettes (tip resistances of 2-4MQ) are filled with amphotericin B (200 ⁇ g/ml) containing internal solution.
  • the internal solution is composed of (mmol/L): 110 K+aspartate, 20 KCL, 1 MgCh, 5 Mg2+ATP, 0.1 Li+GTP, 10 HEPES, 5 Na+phosphocreatine, 0.05 EGTA (pH adjusted to 7.3 with KOH).
  • the cells are treated with lOnM ATX-II (Alomone Labs, Jerusalem, Israel).
  • APD is determined at 90% (APD90) repolarization, excluding cells with AP alternans.
  • Rabbits will receive a single intravenous injection of humanized antibody or vehicle and will be challenged using the methoxamine/dofetilide Carlsson’s model. Under anesthesia (ketamine/xylazine), each rabbit will be infused with methoxamine and dofetilide until the animal progresses to TdP. This challenge will be repeated every 48 to 72 hours for a total of five challenges.
  • Example 6 Assessment of tissue distribution, exposure, and activation of non-cardiac KCNQ1 in rabbits treated with humanized anti-KCNQl Antibody
  • Example 7 Evaluating the effect of forced degradation conditions on the kinetics of humanized anti-KCNQl antibody-mediated binding to the KCNQ1 peptide
  • AEKDAVNESGRVEFGSYADA-Cterm SEQ ID NO: 2. All measurements were performed in triplicates at 25 °C in an assay-specific buffer using 96- well plates with an orbital shake speed of 1,000 rpm. To generate the binding curves, lOnM N-terminally biotinylated KCNQ1 peptide was immobilized on streptavidin-coated biosensor tips for 5 minutes, followed by a baseline equilibration for 5 minutes. The biosensor tips were then submerged in wells containing 0.2-14nM of antibody for 5 min to monitor the formation of the mAb-peptide complex.
  • the observed (or apparent) rate constant of Ab-E binding to the peptide is plotted as a linear function of antibody concentration (0.2-14nM), where the dashed lines represent the 95% confidence interval.
  • Figure 9B and the table below specificity of the Ab-E-peptide interaction was determined using heat-denatured Ab-E as well as human IgGl and mouse IgG2a negative controls, all of which showed non-specific background binding.
  • Results All samples were analyzed to ensure consistency between injections. Resulting chromatograms were integrated based on retention times of the peaks and split into HMWS, Main Peak. Results are reported as %peak area corresponding to the individual peak areas. For Main Peak, %peak areas of monomer and LMW were added. Results are shown in Figures 12B-12D.
  • the size distribution of nine antibodies was determined by Dynamic Light Scattering (DLS) using the UNCLE platform (Unchained Labs), the results of which are provided in Table 9 below.
  • DLS Dynamic Light Scattering
  • UNCLE platform Unchained Labs
  • Z-average diameter was determined based on triplicate runs at a 20 mg/ml concentration under isothermal hold at 37°C for 24 hrs, or after undergoing 3x freeze/thaw cycles followed by an isothermal hold at 37°C for 24 hrs.
  • Binding affinity will be measured using humanized antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-E, Ab-F, and Ab-G and the tetrameric KCNQ1 channel. Structural studies of KCNQ1 in the presence of muFl 1 will be performed using cryo-electron microscopy (methods will be similar to Cell. 2020 January 23; 180(2): 340-347). These data will identify the site on KCNQ1 where Ab-A, Ab-B, Ab-C, Ab-D, Ab-E, Ab-F and Ab-G binds, and the structural rearrangements that take place upon binding (in both the open and closed state).
  • Example 10 Assessment of tissue distribution, exposure, and activation of non-cardiac KCNQ1 in rabbits treated with humanized anti-KCNQl Antibody
  • the tissue cross-reactivity assay was conducted on 1 donor per tissue at 10 and 20 pg/mL after method development on KCNQ1 expressing CHO cells to identify optimal positive immunohistochemistry staining concentration.
  • Staining of Ab-G (comprising amino acid sequences set forth in SEQ ID NOs: 22 and 20) in human tissues included epithelial cells in the pancreas (ducts), prostate (glands) and small intestine (mucosa). Cytoplasmic staining was observed in human cardiomyocytes in the heart and epithelial cells in the pancreas (acini). Cytoplasmic staining was also present in cardiomyocytes in the monkey heart. Epithelial membrane staining was observed in various tissues of all non-clinical species including the adrenal cortex of mini-pigs, dogs, rabbits and guinea pigs, and germinal epithelial cells of all non-clinical animal species.
  • IgGl antibodies can be bound by complement proteins, initiating a cascade that can lead to target cell death in a process known as complement dependent cytotoxicity (CDC), antibodies and Fc- engineered variants (i.e., Ab-G (comprising amino acid sequences set forth in SEQ ID NOs: 22 and 20 and Ab-F (comprising amino acid sequence set forth in SEQ ID NO: 18 and 20) have been assessed in their binding to complement protein Clq.
  • CDC complement dependent cytotoxicity
  • a commercially available AlphaLISA kit was used to measure Clq binding to antibodies. Test articles were plated over a range concentrations spanning from 0.01 ug/mL up to 100 ug/mL. Biotinylated anti-human IgG Gab antibody + streptavidin donor beads mix was added, followed by human Clq, and then anti-Clq acceptor beads. Following incubation, plates were analyzed in a plate reader. The values were then plotted on an XY chart, graphing ALphaLISA signal values against the antibody concentration, and the data was fit to a four-parameter nonlinear regression curve using GraphPad Prism 9.0. Human IgG F(ab’)2 was used as a negative control for Clq binding. Each data point represents the mean of triplicate values. Ab-E exhibits strong binding to Clq. Ab-F and Ab-G do not show binding to Clq.
  • Example 13 FcRn Binding
  • Antibody binding to the neonatal Fc receptor (FcRn) is an important mechanism that enhances the in vivo half-life of an antibody by recycling the antibody and protecting it from lysosomal degradation.
  • the binding of antibodies to FcRn was assessed at 0.01 to 100 pg/mL to determine if Fc-engineering mutations (i.e., Ab-G (comprising amino acid sequences set forth in SEQ ID NOs: 22 and 20) and Ab-F (comprising amino acid sequence set forth in SEQ ID NO: 18 and 20) have an impact in vivo half-life.
  • Fc-engineering mutations i.e., Ab-G (comprising amino acid sequences set forth in SEQ ID NOs: 22 and 20)
  • Ab-F comprising amino acid sequence set forth in SEQ ID NO: 18 and 20
  • a commercially available AlphaLISA kit was used to measure test article binding to FcRn. Test articles were plated over a range of concentrations from 0.01 ug/mL to 100 ug/mL. FnRn was then added, followed by human IgG-conjugated acceptor and donor beads. After incubation, plates were analyzed in a plate reader. The values were then plotted on an XY chart, graphing ALphaLISA signal values against the antibody concentration, and the data was fit to a four-parameter nonlinear regression curve using GraphPad Prism 9.0. Human IgG F(ab’)2 was used as a negative control for FcRn binding. Ab-E and Ab-F exhibit strong binding to FcRn, while Ab-G shows enhanced binding.
  • Ab-F demonstrated binding to FcRn, similarly to Ab-E.
  • Ab-G demonstrated enhanced binding to FcRn compared to Ab-E, with a lower EC50 (IC50) value.
  • Example 14 Functional assessment of humanized anti-KCNQl antibodies in LQT2 patient-derived cells
  • Example 15 Effects of KCNQ1 antibodies on E-4031-induced APD prolongation in Ncytes cardiomyocytes
  • EADs early after depolarizations
  • Action potentials are recorded in cells treated with 60 pg/mL muAb-1 ( Figure 14B) or 60 pg/mL Ab-E ( Figure 14C) followed by E4031 (25 nM) challenge.
  • Statistical analysis One-Way ANOVA with multiple comparisons, Fisher’s ESD test. Results show that Ab-E protected against E4O31 -induced APD prolongation.
  • Results showed that subcutaneously administered Parental Ab-E shortens the rabbit QT interval for at least 27 days at 2 mg/kg. IV-administered Ab-F shortens the rabbit QT interval for ⁇ 4-5 weeks. IV- administered Ab-G shortens the rabbit QT interval for 5+ weeks. Subcutaneously administered Ab-G shortens the rabbit QT interval for ⁇ 5 weeks.
  • the time taken to reach TdP is converted to a cumulative dose of dofetilide infused.
  • New Zealand White rabbits were treated with an intravenous or subcutaneous dose of anti- KCNQ1 monoclonal antibody.
  • Serial blood samples were collected and processed to plasma. Circulating anti-KCNQl antibody concentration was quantitated using mass spectrometry.
  • PK parameters were estimated by non-compartment analysis using Phoenix WinNonlin (version 8.2 Pharsight Corp., Mountain View, California, USA).
  • Mean 11/2 of Ab-G is ⁇ 11.57 days upon excluding 5 mg/kg animals.
  • Mean tl/2 of Ab-F constructs is ⁇ 5-7 days.
  • Mean tl/2 of Ab-E ⁇ 7 days and median tl/2 ⁇ 5.7 days ranging from 1.71-10.4 days
  • Example 21 Assessment of tissue distribution, exposure, and activation of non-cardiac KCNQ1 in rabbits treated with humanized anti-KCNQl Antibody

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Abstract

La présente invention concerne des anticorps humanisés anti-KCNQl et leur utilisation dans le traitement du syndrome du QT long (LQTS).
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