FIELD OF THE DISCLOSURE

[0001] The present application relates to materials and methods for the treatment of Long QT Syndrome.

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

[0002] This application contains, as a separate part of disclosure, a Sequence Listing in computer- readable form (Filename: 58050_SeqListing.xml; 32,151 bytes - XML file created September 1, 2023) which is incorporated by reference herein in its entirety.

BACKGROUND

[0003] Long QT syndrome (LQTS) is responsible for a significant proportion of sudden cardiac deaths

(1). Genetic mutations leading to a loss of function of the cardiac voltage-gated KCNH2 (LQTS type 2 [LQTS2]) or KCNQ1 (LQTS type 1 [LQTS1]) potassium ion (K ⁺ ) channels are the most common causes

(2). As a result, the corresponding repolarizing currents across the KCNH2 (or human Ether-a-go-go

(hERG), Kvll.l) and KCNQ1 (or K _V LQT1, K _v 7.1) channels, and IKS, respectively, are reduced, which prolongs the cardiac repolarization phase. On a surface electrocardiogram (ECG), this delay is reflected by a prolonged QT interval predisposing patients to life-threatening arrhythmias. Current treatment options for LQTS patients include beta-blockers, left cardiac sympathetic denervation, and the implantation of a cardioverter-defibrillator (3).

[0004] Some 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).

[0005] LQTS3 is caused by gain-of-function mutations in the SCN5A-encoded Navi.5 sodium ion (Na ⁺ ) channel.

SUMMARY

[0006] Provided herein are humanized antibodies that bind to KCNQ1 (Potassium Voltage-Gated Channel Subfamily Q Member 1). Murine antibodies that bind to KCNQ1 are disclosed in PCT/IB2022/058286.

[0007] In one aspect, described herein is 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. [0008] In some embodiments, 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.

[0009] In some embodiments, 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.

[0010] In some embodiments, 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.

[0011] In some embodiments, 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.

[0012] In some embodiments, 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.

[0013] In some embodiments, 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.

[0014] In some embodiments, 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.

[0015] In some embodiments, the antibody comprises a heavy chain constant domain (e.g., IgGl, IgG2, IgG3 or IgG4). In some embodiments, the antibody comprises a light chain constant domain. In some embodiments, the heavy chain constant domain is a modified constant domain.

[0016] The disclosure also provides a pharmaceutical composition comprising an antibody described herein and a pharmaceutically acceptable carrier, diluent or excipient.

[0017] Nucleic acids encoding an antibody described herein, as well as vectors and host cells comprising vectors encoding the nucleic acids are also contemplated.

[0018] In another aspect, described herein is a method of treating a subject suffering from long QT syndrome (LQTS) comprising administering the antibody to the subject in an amount effective to treat long QT syndrome. In some embodiments, the long QT syndrome is LQTS1, LQTS2 or LQTS3.

[0019] In some embodiments, the methods described herein optionally further comprise administering a standard of care to the subject for the treatment of long QT syndrome. In some embodiments, the standard of care is a beta-blocker, an implantable cardioverter-defibrillator (ICD), or a left cardiac sympathetic denervation.

[0020] In some embodiments, the subject is also suffering from cardiomyopathy, diabetes, epilepsy or neurological comorbidities.

[0021] In some embodiments, 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.

BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 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.

[0023] Figure 2. 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.

[0024] 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).

[0025] 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).

[0026] Figure 5. 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.

[0027] Figure 6. 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Figure 10 is a graph showing the results of a Clq binding immunoassay.

[0032] Figure 11 is a graph showing the results of a FcRn binding immunoassay. [0033] 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, whereas 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).

[0034] 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).

[0035] 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. E-4031 -induced prolongation of APD90 in Ncytes cardiomyocytes in the absence and presence of KCNQ1 antibody treatment (60 pg/mL) as percentage of change (Figure 14D) and absolute change (Figure 14E). Bars represent mean ± SEM. Statistical analysis: One-Way ANOVA with multiple comparisons, Fisher’s LSD test.

[0036] Figure 15 provides graphs showing the effects of muAb-1 and Ab-E) on Ncytes cardiomyocytes.

[0037] Figures 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.

[0038] Figures 17A and 17B is a graph showing that Ab-E (2 mg/kg) protected against drug-induced QT prolongation and arrhythmia in rabbits.

[0039] Figure 18 are graphs showing the mean plasma concentration of antibodies Ab-E. Ab-F, and Ab- G over time.

DETAILED DESCRIPTION

[0040] 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.

[0041] In one aspect, described herein is a humanized antibody that specifically binds human KCNQ1 (SEQ ID NO: 1). The antibody may be any type of antibody, i.e., immunoglobulin, known in the art. In exemplary embodiments, the antibody is an antibody of class or isotype IgA, IgD, IgE, IgG, or IgM. In exemplary embodiments, the antibody described herein comprises one or more alpha, delta, epsilon, gamma, and/or mu heavy chains. In various embodiments, the antibody is an IgGl, IgG2, or IgG4 antibody. In exemplary embodiments, the antibody described herein comprises one or more kappa or lambda light chains.

[0042] 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 2 x 10 ⁹ M, less than or equal to 3 x 10 ⁹ M, less than or equal to 4 x 10 ⁹ M, less than or equal to 5 x 10 ⁹ M, less than or equal to 6 x 10 ⁹ M, less than or equal to 7 x 10 ⁹ M, less than or equal to 8 x 10 ⁹ M, less than or equal to 9 x 10 ⁹ M, less than or equal to 1 x 10 ¹⁰ M, less than or equal to 2 x 10 ¹⁰ M, less than or equal to 3 x 10 ¹⁰ M, less than or equal to 4 x 10 ¹⁰ M, less than or equal to 5 x 10 ¹⁰ M, less than or equal to 6 x 10 ¹⁰ M, less than or equal to 7 x 10 ¹⁰ M, less than or equal to 8 x 10 ¹⁰ M, less than or equal to 9 x 10 ¹⁰ M, less than or equal to 1 x 10 ¹¹ M, less than or equal to 2 x 10 ¹¹ M, less than or equal to 3 x 10 ¹¹ M, less than or equal to 4 x 10 ¹¹ M, less than or equal to 5 x 10 ¹¹ M, less than or equal to 6 x 10 ¹¹ M, less than or equal to 7 x 10 ¹¹ M, less than or equal to 8 x 10 ¹¹ M, less than or equal to 9 x 10 ¹¹ M, less than or equal to 1 x 10 ¹² M, less than or equal to 2 x 10 ¹² M, less than or equal to 3 x 10 ¹² M, less than or equal to 4 x 10 ¹² M, less than or equal to 5 x 10 ¹² M, less than or equal to 6 x 10 ¹² M, less than or equal to 7 x 10 ¹² M, less than or equal to

8 x 10 ¹² M, or less than or equal to 9 x 10 ¹² M. It will be appreciated that ranges having the values above as end points is contemplated in the context of the disclosure. For example, 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

9 x 10 ¹² M or an affinity of 1 x 10 ⁹ to about 9 x 10 ¹² . In various embodiments, the KCNQ1 is human KCNQ1 (Genbank Accession No. NP_000209, 676 amino acids, potassium voltage-gated channel subfamily KQT member 1 isoform 1).

[0043] In various embodiments, 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. [0044] In some or any embodiments, 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. 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. 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.

[0045] In some or any embodiments, 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. 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 and retains the CDR amino acid sequences set out in SEQ ID NOs: 10-12.

[0046] In some embodiments, 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.

[0047] In some or any embodiments, 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. 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 and retains the CDR amino acid sequences set out in SEQ ID NOs: 13 and 14 and the LCDR2 amino acid sequence “WAS”.

[0048] In some or any embodiments, 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. 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 and retains the CDR amino acid sequences set out in SEQ ID NOs: 13 and 14 and the LCDR2 amino acid sequence “WAS”.

[0049] In some or any embodiments, 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. 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 and retains the CDR amino acid sequences set out in SEQ ID NOs: 13 and 14 and the LCDR2 amino acid sequence “WAS”.

[0050] In some or any embodiments, 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. 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 and retains the CDR amino acid sequences set out in SEQ ID NOs: 13 and 14 and the LCDR2 amino acid sequence “WAS”.

[0051] In some embodiments, 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.

[0052] 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 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 5 (Antibody Ab-A). [0053] 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 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 5 (Antibody Ab-B).

[0054] 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 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 8 (Antibody Ab-C).

[0055] 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 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 6 (Antibody Ab-D).

[0056] 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 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 9 (Antibody Ab-E).

[0057] In some or any embodiments, 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.

[0058] In some or any embodiments, 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.

[0059] In some or any embodiments, 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.

[0060] In some or any embodiments, 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.

[0061] In some or any embodiments, 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.

[0062] In some or any embodiments, 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.

[0063] In some or any embodiments, 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.

[0064] In some or any embodiments, 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.

[0065] In some or any embodiments, 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.

[0066] In some embodiments, 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. [0067] In some embodiments, 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.

[0068] In some embodiments, 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. (Ab-F)

[0069] In some embodiments, 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. (Ab-G)

[0070] In some embodiments, 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.

[0071 ] 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. For ease of reference, 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 ;. scFv, dsFv, scFv-Fc, bispecific diabodies, trispecific triabodies, minibodies, a fragment of IgNAR (e.g., V-NAR), a fragment of hdgG (e.g., VhH), bis-scFvs, fragments expressed by a Fab expression library, and the like. In exemplary aspects, 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, however, are not limited to these exemplary types of antibody fragments. In exemplary aspects, antigen binding fragment is a Fab fragment. In exemplary aspects, 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.

[0072] 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.

[0073] Methods of Antibody or Antigen Binding Fragment Production

[0074] 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. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (Nature 256: 495-497, 1975), the human B-cell hybridoma technique (Kosbor et al., Immunol Today 4:72, 1983; Cote et al., Proc Natl Acad Sci 80: 2026-2030, 1983) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96, (1985). Alternatively, other methods, such as EBV-hybridoma methods (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), and Roder et al., Methods Enzymol., 121, 140-67 (1986)), and bacteriophage vector expression systems (see, e.g., Huse et al., Science, 246, 1275-81 (1989)) are known in the art. Further, methods of producing antibodies in non-human animals are described in, e.g., U.S.

Patents 5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002/0197266 Al). 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.

[0075] Phage display also can be used to generate the antibody of the present disclosures. In this regard, 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). 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. The techniques described in U.S. Patent No. 5,780,279; U.S. Patent No. 5,821,047; U.S. Patent No. 5,824,520; U.S. Patent No. 5,855,885; U.S. 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.

[0076] 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.

[0077] 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)).

[0078] Methods of testing antibodies for the ability to bind to an epitope of KCNQ1 , regardless of how the antibodies are produced, 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).

[0079] 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.

[0080] A single-chain variable region fragments (scFv), 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). Similarly, 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)).

[0081] Recombinant antibody fragments, e.g., scFvs, can also be engineered to assemble into stable multimeric oligomers of high binding avidity and specificity to different target antigens. Such diabodies (dimers), triabodies (trimers) or tetrabodies (tetramers) are well known in the art, see e.g., Kortt et al., Biomol Eng. 2001 18:95-108, (2001) and Todorovska et al., J Immunol Methods. 248:47-66, (2001).

[0082] 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.

[0083] 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). In some embodiments, 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,

292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 312, 315, 318, 320, 322, 324, 326, 327, 329, 330,

331, 332, 333, 334, 335, 337, 338, 339, 340, 345, 356, 359, 360, 361, 373, 376, 379, 382, 383, 388, 389,

398, 400, 409, 414, 416, 419, 422, 430, 434, 435, 437, 438, 439, 440, or 442.

[0084] 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]:

[0085] S239D/I332E

[0086] S239D/A330S/I332E

[0087] S239D/A330L/I332E

[0088] S298A/D333A/K334A

[0089] P247I/A339D

[0090] P247I/A339Q

[0091] D280H/K290S

[0092] D280H/K290S/S298D

[0093] D280H/K290S/S298V

[0094] F243L/R292P/Y300L

[0095] F243L/R292P/Y300L/P396L

[0096] F243L/R292P/Y300L/V305I/P396L

[0097] G236A/S239D/I332E

[0098] K326A/E333A

[0099] K326W/E333S [0100] K290E/S298G/T299A

[0101] K290N/S298G/T299A

[0102] K290E/S298G/T299A/K326E

[0103] K290N/S298G/T299A/K326E

[0104] K334V

[0105] L235S+S239D+K334V

[0106] Q311M+K334V

[0107] S239D+K334V

[0108] F243V+K334V

[0109] E294L+K334V

[0110] S298T+K334V

[0111] E233L+Q311M+K334V

[0112] L234I+Q311M+K334V

[0113] S298T+K334V

[0114] A330M+K334V

[0115] A330F+K334V

[0116] Q311M+A330M+K334V

[0117] Q311M+A330F+K334V

[0118] S298T+A330M+K334V

[0119] S298T+A330F+K334V

[0120] S239D+A330M+K334V

[0121] S239D+S298T+K334V

[0122] L234Y+K290Y+Y296W

[0123] L234Y+F243V+ Y296W

[0124] L234Y+E294L+ Y296W

[0125] L234Y + Y296W

[0126] K290Y + Y296W [0127] In some embodiments, 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]:

[0128] N297A (IgGl)

[0129] L234A/L235A (IgGl)

[0130] V234A/G237A (IgG2)

[0131] L235 A/G237 A/E318 A (IgG4)

[0132] H268Q/V309L/A330S/A331 S (IgG2)

[0133] C220S/C226S/C229S/P238S (IgGl)

[0134] C226S/C229S/E233P/L234V/L235A (IgGl)

[0135] L234F/L235E/P331S (IgGl)

[0136] S267E/L328F (IgGl)

[0137] 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. These include recombinant expression in certain mammalian cell lines including a FUT8 knockout cell line, variant CHO line Eecl3, rat hybridoma cell line YB2/0, a cell line comprising a small interfering RNA specifically against the FUT8 gene, and a cell line coexpressing [3-1,4- N-acetylglucosaminyltransferase III and Golgi [3-mannosidase II. Alternatively, the Fc-containing molecule may be expressed in a non-mammalian cell such as a plant cell, yeast, or prokaryotic cell, e.g., E. coli. Thus, in certain embodiments, a composition comprises an antibody having reduced fucosylation or lacking fucosylation altogether.

[0138] In some embodiments, the humanized antibodies have an Fc having one or more of the following substitutions (IgGl): N297D N297G N297A N297Q

E234A and E235A

E234A, and E235A, and P329D

M252Y, S254T and T256E

S228P S228P and L235E

S228P and P329G

S228P, L235E and P329G

L234F, L235E and P331S

L234F, L235E, P331S and M252Y, S254T and T256E

[0139] 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. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (Nature 256: 495-497, 1975), the human B-cell hybridoma technique (Kosbor et al., Immunol Today 4:72, 1983; Cote et al., Proc Natl Acad Sci 80: 2026-2030, 1983) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96, (1985). Alternatively, other methods, such as EBV-hybridoma methods (Haskard and Archer, J. Immunol.

Methods, 74(2), 361-67 (1984), and Roder et al., Methods Enzymol., 121, 140-67 (1986)), and bacteriophage vector expression systems (see, e.g., Huse et al., Science, 246, 1275-81 (1989)) are known in the art. Further, methods of producing antibodies in non-human animals are described in, e.g., U.S. Patents 5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002/0197266 Al). 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.

[0140] Phage display also can be used to generate the antibody of the present disclosures. In this regard, 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). 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. The techniques described in U.S. Patent No. 5,780,279; U.S. Patent No. 5,821,047; U.S. Patent No. 5,824,520; U.S. Patent No. 5,855,885; U.S.

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.

[0141] 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.

[0142] Methods for generating humanized antibodies are well known in the art and are described in detail in, for example, Janeway et al., supra, U.S. Patent Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No. 0239400 Bl, and United Kingdom Patent No. 2188638. Humanized antibodies can also be generated using the antibody resurfacing technology described in U.S. Patent No. 5,639,641 and Pedersen et al., J. Mol. Biol, 235, 959-973 (1994). A preferred chimeric or humanized antibody has a human constant region, while the variable region, or at least a CDR, of the antibody is derived from a non-human species. Methods for humanizing non-human antibodies are well known in the art. (See, e.g., U.S. Patent Nos. 5,585,089, and 5,693,762.)

[0143] 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). For example, knob in hole changes can refer to a T366W (“knob”) change, as well as T366S, L368A, and Y407V (“hole”) alterations. Additional substitutions may provide opportunities for C-C interaction between the chains, e.g., S354C and Y349C (cysteine 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.

[0144] 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 are well known in the art and 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. [0145] 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. Cell lines of particular preference are selected through determining which cell lines have high expression levels and produce antibodies with KCNQ1 -binding properties.

[0146] In various embodiments, 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.

[0147] Also contemplated is a vector comprising the nucleic acid encoding the antibody. In various embodiments, the nucleic acid encoding a heavy chain variable region and light chain variable region are expressed on the same vector or different vectors.

[0148] Further provided is a host cell comprising a nucleic acid encoding a humanized antibody heavy and/or light chain variable region or vector expressing said nucleic acid. In some embodiments, the host cell is an eukaryotic cell.

[0149] Detection Methods

[0150] It is sometimes desirable to detect the presence or measure the amount of KCNQ1 in a sample. In this regard, the disclosure provides a method of using the humanized antibody or fragment thereof described herein to measure the amount of KCNQ1 in a sample. To determine a measurement of KCNQ1 , 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. For example, the humanized antibody can be quantitatively measured if it has a detectable label, or a secondary antibody can be used to quantify the immunocomplex.

[0151] In some embodiments, the biological sample comprises a tissue sample, a cell sample, or a biological fluid sample, such as blood, saliva, serum, or plasma.

[0152] 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. 3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The 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.

[0153] 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.

[0154] In some embodiments, 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.

[0155] Detection of the presence or absence of a KCNQl-antibody complex can be achieved by using any method known in the art. For example, the transcript resulting from a reporter gene transcription assay of a KCNQ1 peptide interacting with a target molecule (e.g., antibody) typically encodes a directly or indirectly detectable product (e.g., P-galactosidase activity and luciferase activity). For cell free binding assays, one of the components usually includes, or is coupled to, a detectable label. A wide variety of 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.

[0156] A variety of methods can be used to detect the label, depending on the nature of the label and other assay components. For example, the 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.

[0157] Pharmaceutical Compositions

[0158] Pharmaceutical compositions comprising a humanized KCNQ1 antibody or antigen binding fragment thereof described herein are also contemplated. In some embodiments, 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. In such embodiments, 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, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counter-ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. See, REMINGTON’S PHARMACEUTICAL SCIENCES, 18" Edition, (A. R. Genrmo, ed.), 1990, Mack Publishing Company.

[0159] 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. For example, 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. In specific embodiments, pharmaceutical 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. In certain embodiments, 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. Further, in some embodiments, the antibody or (antigen binding fragment thereof) may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0160] The pharmaceutical 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.

[0161 ] When parenteral administration is contemplated, 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. In certain embodiments, implantable drug delivery devices may be used to introduce the desired antibody (or antigen binding fragment thereof).

[0162] Additional pharmaceutical 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. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra) or poly-D(-)-3-hydroxybutyric acid (European Patent Application Publication No. EP133988). 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.

[0163] Embodiments of the humanized antibody formulations can further comprise one or more preservatives.

[0164] Administration of the 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.

[0165] Dosage [0166] In some embodiments, 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. For example, 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). In some embodiments, 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).

[0167] It may be advantageous to administer multiple doses of the antibody or antigen binding fragment at a regular interval, depending on the therapeutic regimen selected for a particular subject. In some embodiments, 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). In this regard, 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.

[0168] In various embodiments, 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). For example, 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 least about 950 mg or up to about 1,000 mg of antibody. 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.

[0169] In some embodiments, 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). For example, 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 31 mg/kg, at least about 32 mg/kg, at least about 33 mg/kg, at least about 34 mg/kg, at least about 35 mg/kg, at least about 36 mg/kg, at least about 37 mg/kg, at least about 38 mg/kg, at least about 39 mg/kg, at least about 40 mg/kg, at least about 41 mg/kg, at least about 42 mg/kg, at least about 43 mg/kg, at least about 44 mg/kg, at least about 45 mg/kg, at least about 46 mg/kg, at least about 47 mg/kg, at least about 48 mg/kg, at least about 49 mg/kg, at least about 50 mg/kg, at least about 55 mg/kg, at least about 60 mg/kg, at least about 65 mg/kg, at least about 70 mg/kg, at least about 75 mg/kg, at least about 80 mg/kg, at least about 85 mg/kg, at least about 90 mg/kg, at least about 95 mg/kg, or up to about 100 mg/kg. 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.

[0170] Methods of Treatment

[0171] In another aspect, described herein is a method of treating a subject suffering from long QT syndrome (LQTS) comprising administering the humanized antibody or pharmaceutical composition described herein to the subject in an amount effective to treat long QT syndrome.

[0172] In some embodiments, 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.

[0173] In some embodiments, the subject is also suffering from cardiomyopathy, diabetes, epilepsy or neurological comorbidities. In some embodiments, 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.

[0174] In some embodiments, the subject is suffering from LQT3. In some embodiments, the subject has acquired long QT. In some embodiments, the subject has acute QT prolongation.

[0175] In some embodiments, 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.

[0176] In some embodiments, administering the humanized antibody does not affect KCNQ1 channel expression in the subject.

[0177] In some embodiments the antibody has no detectable or minimal off-target effects, e.g. epilepsy, neuropsychiatric comorbidities, diabetes mellitus or impaired glucose tolerance, thyroid disorder.

[0178] Combination Therapy

[0179] The humanized antibodies disclosed here can be administered alone or optionally in combination with other therapeutic agents useful for the treatment of LQTS. Thus, 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®).

[0180] In some embodiments, the antibody is administered in combination with an agent that would otherwise prolong the QT interval of patients.

[0181] Kits

[0182] Once a pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration. The invention also provides kits for producing a single-dose administration unit. The kits of the disclosure may each contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes) are provided.

[0183] The following Examples are provided to further illustrate aspects of the disclosure, and are not meant to constrain the disclosure to any particular application or theory of operation.

EXAMPLES

Example 1 - Effect of humanized KCNQ1 antibodies on KCNQ1/KCNE1 current density

[0184] Materials and Methods [0185] Cell culture: tsA201 cells (transformed human embryonic kidney 293 cells) were grown in modified Eagle’s medium (MEM) supplemented with 10% fetal calf serum and 100 μg/ml penicillin, 100 μg/ml streptomycin, and 0.25 100 μg/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. The day before transfection, cells were washed with MEM, treated with trypsin/EGTA for one minute and plated on 25 mm coverslips. KCNQ1, KCNE1 and GFP cDNAs (0.1+0.3pg) were transfected using lipofectamine 2000.

[0186] 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.

[0187] 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.

[0188] Table 2.

[0189] A current-voltage (I-V) protocol consisting of a 4-second step protocol with pulses from -80 mV to +80 mV, followed by a repolarizing step to -40 mV for 2 seconds was applied. Holding potential was - 90 mV and interpulse interval was 15 seconds.

[0190] 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.

[0191] Table d.

[0192] Analysis: Microsoft Excel (version 16.36) and GraphPad Prism (version 9.1) were used to analyze the data. Conductance-voltage (G-V) relationships were generated by normalizing instantaneous tail current amplitude at each step voltage to the maximum tail current amplitude(G/Gmax). Voltage dependence of channel activation was fitted with a standard single-component Boltzmann equation of the form:

[0193] Y= Bottom + (Top-Bottom)/1+ exp (V 1/2-V/k)

[0194] Where V is the potential, V 1/2 is the voltage where channels exhibit half-maximal activation (between bottom and top), and 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.

[0195] The experiment was repeated, and activation V1/2 and slope value (k-f actor) in the absence and presence of 60 pg/mE of the test antibodies. Results are shown below in Table 5.

[0196] Results:

[0197] Current density was increased in the presence of muAb-1, Ab-C, and Ab-E at a concentration of 30 ug/mL (Figure 1) and 60 ug/mL (Figure 2), and also for Ab-A at 60 ug/mL (Figure 2). At 30 ug/mL muAb-1 increased current density over control at voltages between -20 mV and + 80 mV (Figure 3A). Importantly, the increase in current density at membrane potentials more positive than -20 mV corresponds

T1 to the physiologically relevant potentials at which IKs channel is open. (Jesperson et al., Physiology 2005 20:6, 408-416). At 60 ug/mL, Ab-E, Ab-C, and Ab-A exhibited increases in current density (Figure 3A and Figures 5 and 6). Ab-E shows a dose-dependent increase in current density.

[0198] At 30 ug/mE, muAb-1 significantly shifted the voltage-dependence of activation to more negative potentials, with a V1/2 of 9.6 +/- 10.2 mV vs 24.4 +/- 5.9 mV for control (Figure 4B, Table 4). At 60 ug/mL a significant shift of the voltage-dependence of activation of the IK _S channel to more negative potentials was observed for muAb-1, Ab-E, Ab-D, and Ab-A (Figure 4B; Table 4)

[0199] Table 4. Activation Vi/2and slope value (k-factor) in the absence (control) and in the presence of 30 pg/mL of various antibodies.

[0200] Table 5. Activation V1/2 and slope value (k-factor) in the absence (control) and in the presence of 60 pg/mL of various antibodies.

[0201] IKS channel deactivation was slowed in the presence of muFl 1 and, to a lesser extent, Ab-E at 30 ug/mL (Figure 7A). At 60 ug/mL, channel deactivation was slowed by muFl 1, Ab-E, and Ab-C (Figure 7B). [0202] These results show that the parental mouse monoclonal antibody against KCNQ1 (muAb-1) and some of its humanized derivatives (namely Ab-E and Ab-C) increased ion flux in HEK293 cells transiently transfected to express the human IKs channel.

Example 2 - Binding Affinity of Humanized Anti-KCNQl Antibodies

[0203] 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.

[0204] Results:

[0205] muAb-1 and chAb-2 interacted with the KCNQ1 peptide with the highest affinity (KD = ~4nM), whereas each of the humanized antibodies bound with KDs in the 6-34 nM range as shown below in Table 6 and Figure 8.

[0206] Table d.

[0207] All tested antibodies interacted specifically with the KCNQ1 peptide with relatively high affinities, in the low to intermediate nanomolar range. § Example 3 - Functional assessment of humanized anti-KCNQl antibodies in LQT2 patient-derived cells

[0208] The following experiment will be performed in order to assess the ability of humanized antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-E, Ab-F, and Ab-G to shorten action potential duration and reduce early after depolarization, beating arrhythmias, and beating arrest in LQT2 patient-derived and pharmacologically-induced iPSC-CMs .

[0209] In brief, glass coverslips are coated with Matrigel (Corning) and placed into each well of a 24-well plate. Cardiomyocytes are diluted with plating medium to reach a density of 30,000-35,000 cells/well. Cardiomyocytes are maintained in a cell culture incubator at 37 °C, 5% CO2 for the duration of the experiment (starting at least 7 days post plating). Maintenance Medium is replaced every other day.

[0210] For patch clamp experiments, 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 60μg/ml, 24h). Borosilicate glass pipettes (tip resistances of 2-4MQ) are filled with amphotericin B (200μg/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). To induce LQTS2 in hiPSC-CMC, 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

[0211] The following experiment will be performed in order to assess the ability humanized antibodies Ab-A, Ab-B, Ab-C, Ab-D and Ab-E to shorten action potential duration and reduce early after depolarization, beating arrhythmias, and beating arrest in LQT3 patient-derived and pharmacologically- induced iPSC-CMs.

[0212] In brief, glass coverslips are coated with Matrigel (Corning) and placed into each well of a 24-well plate. Cardiomyocytes are diluted with plating medium to reach a density of 30,000-35,000 cells/well. Cardiomyocytes are maintained in a cell culture incubator at 37 °C, 5% CO2 for the duration of the experiment (starting at least 7 days post plating). Maintenance Medium is replaced every other day.

[0213] For patch clamp experiments, 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 60μg/ml, 24h). Borosilicate glass pipettes (tip resistances of 2-4MQ) are filled with amphotericin B (200μg/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). For inducing LQT3 in hiPSC-CMC, the cells are treated with lOnM ATX-II (Alomone Labs, Jerusalem, Israel). APD is determined at 90% (APD90) repolarization, excluding cells with AP alternans.

Example 5 - In vivo duration of effect of humanized anti-KCNQl antibodies

[0214] The following experiment will be performed in order to examine the duration of effect of humanized antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-E, Ab-F, and Ab-G in shortening the baseline QT interval of rabbits, and in preventing QT prolongation and arrhythmias.

[0215] 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

[0216] The following experiment will be performed in order to determine the level of humanized antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-E, Ab-F, and Ab-G in rabbit tissues, and to assess glucose levels as a surrogate for non-cardiac KCNQ1 activation, following a single dose of humanized antibody. Plasma and tissue exposure are measured by ELISA and/or mass spectometry. In addition, assessments are made using histology.

Example 7 - Evaluating the effect of forced degradation conditions on the kinetics of humanized anti-KCNQl antibody-mediated binding to the KCNQ1 peptide

[0217] The following experiment was performed to evaluate the kinetics of humanized anti-KCNQl antibody binding to the 20 aa KCNQ1 target sequence (Nterm-Biotin-(CH2O)4-

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. As shown in 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. As shown in 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.

[0218] Table ?.

[0219] The experiment described above was repeated with Ab-F and Ab-G antibodies, results of which are shown in Figure 12A. In addition, Ab-F and Ab-G were subjected to a variety of forced degradation conditions to compare the observed (or apparent) binding rate constants and content of high molecular weight species (%HMWS) were assessed by Size-Exclusion Ultrahigh Pressure Liquid Chromatography (SEC-UPLC). See Table 8 below. Samples were diluted to 5 mg/mL using formulation buffer prior to analysis. Samples (25 pg) were then injected onto a SEC column (Waters Acquity BEH SEC 4.6 mm x 150 mm). Sample was eluted isocratically using mobile phase consisting of 10 mM Sodium Phosphate, 0.5 M Sodium Chloride pH 6.2.

[0220] 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.

[0221] Table 8.

[0222] Next, 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. For each antibody, the Z-average diameter (nm) 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.

[0223] Table 9.

[0224] Next, the thermal melting profiles for Ab-E, Ab-F and Ab-G weres monitored by differential scanning fluorimetry (DSF) using the UNCLE platform (Unchained Labs). Each antibody was formulated at a concentration of Img/mL with lOx SYPRO® Orange fluorescent dye. Nine pL was loaded into a Uni and subjected to a thermal ramp from 25 °C to 95 °C, with a ramp rate of 1.0 °C/minute and excitation at 473 nm. The data were analyzed by the UNCLE software, which calculated the area under the curve between 510-680 nm to determine the inflection point of the transition curve (Tm). All measurements were performed in triplicate. Results are shown in Figure 13.

Example 8 - Evaluating mechanism of action

[0225] The following experiment will be performed to determine the mechanism by which humanized antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-E, Ab-F, and Ab-G elicit agonistic activity on KCNQ1.

[0226] 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). Various fragments (such as FAB, (FAB’)2) of Ab-A, Ab-B, Ab-C, Ab-D, Ab-E, Ab-F and Ab-G will be generated using standard methods to assess whether monovalent binding is sufficient or if multivalent binding is required to elicit functional effect.

Example 9 - Effect of mutations in the IgGl framework

[0227] The following experiment will be performed in order to evaluate whether various mutations in the IgGl framework of humanized antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-E, Ab-F, and Ab-G affect the biophysical and functional properties of the antibody (such as binding affinity, cellular activity, in vivo activity).

[0228] Upon production of IgGl variants, a series of experiments will be performed to evaluate the effect of specific mutations on complement binding/activation of complement; binding to FcG receptors; antibody-dependent cellular cytotoxicity; antibody-dependent cellular phagocytosis; binding affinity to KCNQ1 peptide and/or protein; functional activation of KCNQ1 via electrophysiology in HEK293 cells expressing KCNQ1/KCNE1, and in iPSC-CMs (drug-induced and EQT2/EQT3-patient derived); in vivo effect on shortening baseline QT interval and in conferring protection against drug-induced QT prolongation/TdP in rabbits or similar models (which may include, but are not limited to, guinea pigs, NHPs, dogs, pigs). A variety of standard binding and/or cellular and/or functional assays may be used for these assessments.

Example 10 - Assessment of tissue distribution, exposure, and activation of non-cardiac KCNQ1 in rabbits treated with humanized anti-KCNQl Antibody

[0229] 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.

Example 11 - Tissue Cross Reactivity Study

[0230] The data provided in this Example demonstrates that Antibody G is cross-reactive across all animal species. The differences in staining between human and the nonclinical species could be due to individual donor variation, section to section differences or might represent true species differences. Results of membrane staining is summarized in Table 10 below:

Example 12 - Clq Binding

[0231] Since 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.

[0232] 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.

[0233] Binding of Ab-F and Ab-G to Clq was assessed at 0.01 to 100 pg/mL. As shown in Figure 10, the parental antibody (Ab-E) demonstrated high receptor binding for Clq clear dose response curve was observed. Neither Ab-F nor Ab-G showed any binding to Clq.

Example 13 - FcRn Binding [0234] 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.

[0235] 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.

[0236] 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

[0237] The following experiment was performed to examine the ability of anti-KCNQl monoclonal antibodies to shorten the action potential duration (APD) in normal IPSC-cardiomyocytes. All measurements were made at 35-37°C at a stimulation rate of 1 Hz. A: APD90 (A), APD50 (B), maximum diastolic potential (MDP) (C) and action potential amplitude (APA) (D) in the absence (control) and presence of KCNQ1 antibody treatment. Each point represents an individual cell measurement and bars represent mean ± SEM. Statistical analysis: One-Way ANOVA with Dunnett’s multiple comparisons test. P values (numbers) are indicated on each graph (See Figure 15).

[0238] As shown in Table 11 below, Ab-E significantly shortens the APD at 50% repolarization (APD50). Shortening of APD at 90% repolarization (APD90) is trending towards significance.

[0239] Table 11.

Example 15 - Effects of KCNQ1 antibodies on E-4031-induced APD prolongation in Ncytes cardiomyocytes [0240] 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. Action potentials recorded from a control cell (no antibody treatment) in the presence of E-4031 (25 nM), and are shown in Figure 14 A. In this cell, AP is prolonged and early after depolarizations (EADs) are generated. 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. E-4031 - induced prolongation of APD90 in Ncytes cardiomyocytes in the absence and presence of KCNQ1 antibody treatment (60 pg/mL) as percentage of change (Figure 14D) and absolute change (Figure 14E). Bars represent mean ± SEM. Statistical analysis: One-Way ANOVA with multiple comparisons, Fisher’s ESD test. Results show that Ab-E protected against E4O31 -induced APD prolongation.

Example 16 - Effect of anti-KCNQl monoclonal antibodies on the baseline QT interval in vivo

[0241] The following experiment was performed to assess the effect of anti-KCNQl monoclonal antibodies on the baseline QT interval of rabbits. Briefly, New Zealand white rabbits were implanted with telemetry probes. Rabbits were treated either with Ab-E (Figures 16A and 16B), Ab-F (Figure 16C), or Ab-G (Figures 16D and 16E), administered either (i) intravenously (Figures 16A, 16C, and 16D) or (ii) subcutaneously (Figures 16B and 16E). ECG recordings were taken daily, and the change in QT interval from baseline (pre-dose) was plotted. Results show that anti-KCNQl monoclonal antibodies shorten the QT interval when given intravenously or subcutaneously.

[0242] 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.

Example 17 - Effect of anti-KCNQl monoclonal antibody on drug-induced QT prolongation and arrhythmia

[0243] The following experiment was performed to determine if a humanized anti-KCNQl monoclonal antibody can protect against drug-induced QT prolongation and arrhythmia in vivo. Two rabbits were prescreened for sensitivity to arrhythmias (torsades de pointes; TdP) via Carlsson challenge* prior to receiving a 2 mg/kg intravenous dose of Ab-E. Carlsson challenge was applied again 4 days post-dose (approximate time of peak effect on baseline QT interval shortening). Carlsson challenge: IV infusion of al -adrenergic receptor agonist (methoxamine) + hERG channel blocker (dofetilide) for 30 min. The time taken to reach TdP is converted to a cumulative dose of dofetilide infused. The change in QT interval following Carlsson model induction (i.e. dofetilide infusion), and the dose of dofetilide administered that led to an arrhythmic event, were plotted.

[0244] Results: [0245] At baseline, 380 pg and 667 pg of dofetilide was required to induce arrhythmia in Rabbit 77 and 79, respectively. Four days after a 2 mg/kg IV dose of Ab-E was administered, Rabbit 77 did not exhibit an arrhythmic event upon Carlsson model induction, while Rabbit 79 required 1350 pg of dofetilide (i.e. twice the dose at baseline) to induce arrhythmia. Therefore, Ab-E appears to have conferred protection against drug-induced Torsades de Pointes. Ab-E treatment conferred a 100 ms protection from drug- induced QT prolongation in Rabbit 77, and a 30 ms protection in Rabbit 79. See Figures 17A and 17B.

Example 18 - Pharmacokinetic analysis

[0246] 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).

[0247] Results are shown in Tables 12 and 13 below:

[0248] Dose linearity was observed for all three mAbs from 0.67 mg/kg to 5 mg/kg.

[0249] IV Mode of Administration: 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

[0250] SC Mode of Administration: Median Tmax of Ab-G is ~4 days ranging from 4-11 days. Median Tmax of Ab-E was ~2 days ranging from 2-4 days. SC dosing with Ab-F was not performed. Sudden decline in plasma concentration due to ADA observed in certain animals affected estimation of PK parameters in those animals.

[0251] Additional results are shown in Figure 18. Excluding 5mg/kg group, Ab-G antibody shows slightly longer tl/2 in IV dose groups.

Table 13. PK Parameter Estimation of parental antibody Ab-A.

SUBSTITUTE SHEET (RULE 26) Example 19 - Off target binding analysis

[0252] An off-target screening panel of 6105 full-length human plasma membrane proteins, secreted and cell surface-tethered human secreted proteins plus a further 400 human heterodimers proteins was conducted to determine if the monoclonal antibodies (i.e., Ab-E and Ab-G) bind to other targets.

[0253] Results:

[0254] Parental Ab-E and Ab-G showed significant specific interactions with the primary target KCNQ1 and did not exhibit binding to any other ion channel. Amongst 6105 proteins screened, the antibodies only showed a specific interaction with two other proteins after fixation only: TSPAN8 (tetraspanin 8) and DBH (dopamine beta hydroxylase).

Example 20 - High Concentration Viscosity Data for Ab-F and Ab-G

[0255] Ab-F and Ab-G samples were concentrated to -100 mg/mL and the viscosities of the samples were measured at 20°C as a function of shear rate (up to 3800 1/s shear rate). M-VROC from RheoSense was used to measure the viscosity of the samples VROC technology which measures dynamic viscosity of a sample over a wide dynamic range of operation, in this case, shear rate.

[0256] Results:

[0257] Increase in shear rate up to 3800 1/s did not affect viscosity of the samples (Newtonian fluid). The viscosity values are an average of 3 runs. No difference in viscosity was observed between Ab-F and Ab- G. Neither of Ab-F and Ab-G were viscous at -100 mg/mE.

Example 21 - Assessment of tissue distribution, exposure, and activation of non-cardiac KCNQ1 in rabbits treated with humanized anti-KCNQl Antibody

[0258] The following experiment will be performed in order to determine the level of humanized antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-E, Ab-F, and Ab-G to assess glucose levels as a surrogate for non-cardiac KCNQ1 activation, following a single dose of humanized antibody.

[0259] The effect of antibody treatment on rabbit blood glucose level was measured using a glucose meter. Results are shown in Figures 19A-19C. IV administration of Ab-E, and subcutaneous administration of Ab-F and Ab-G, do not have an effect on blood glucose levels.

[0260] References: [0261] 1. Barhanin J, Lesage F, Guillemare E, Fink M, Lazdunski M, Romey G. KvLQTl and IsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature. 1996; 384:78-i80.

[0262] 2. Sanguinetti M.C.C.M, Curran M.E, Keating M.T. Coassembly of KvLQTl and minK IsK proteins to form cardiac IKs potassium channel. Nature. 1996; 384:80-83.

[0263] 3. Hund T.J, Rudy Y. Rate dependence and regulation of action potential and calcium transient in a canine cardiac ventricular cell model. Circulation. 2004;110: 3168-3174

[0264] 4. Sanguinetti M.C. Dysfunction of delayed rectifier potassium channels in an inherited cardiac arrhythmia. Ann N Y Acad Sci. 1999; 868: 406-13.

[0265] 5. Chen Y.H, Xu S.J, Bendahhou S, Wang Y, Xu W.Y, Jin H.W, Sun H, Su X.Y, Zhuang Q.N, Yang Y.Q, Li Y.B, Liu Y, Xu H.J, Li X.F, Ma N, Mou C.P, Chen Z, Barhanin J, Huang W. KCNQ1 gain- of-function mutation in familial atrial fibrillation. Science. 2003; 299: 251-4.