Autoantibodies against the NaV1.5 channel as a biomarker for the diagnosis of Brugada syndrome.

Abstract

This invention relates to autoantibodies against NaV1.5 channels and associated isoforms, their use as biomarkers for the diagnosis of Brugada syndrome in humans, and methods relating to the detection of this newly discovered form of autoimmunity and therapeutic approaches targeting it.

Description

Technical Field

[0001] The present invention relates to autoantibodies against NaV1.5 channels and related isoforms, their use as biomarkers for the diagnosis of Brugada syndrome in humans, and related methods for detecting this newly discovered form of autoimmunity and treatment approaches targeting it.

Background Art

[0002] Brugada syndrome (BrS) is a hereditary arrhythmogenic disease with an increased risk of sudden cardiac death (SCD), accounting for 5 - 40% of SCD cases in individuals under 40 years old [1]. This syndrome typically involves cardiac arrest or syncope and occurs in the 30s and 40s [2][3], but most patients are asymptomatic with a normal heart structure and are usually diagnosed incidentally.

[0003] This is diagnosed by the presence of coved - type ST segment elevation in right - chest leads on an electrocardiogram (ECG), which can occur spontaneously or after an induced drug test with intravenous sodium channel blockers [4]. Some patients experience cardiac arrest or syncope in their 30s and 40s, but most patients are asymptomatic [2]. Brugada syndrome is a complex disorder influenced by both genetic and non - genetic factors [5,6], involving mutations in over 23 genes encoding sodium, potassium, and calcium channels, as well as proteins involved in their transport [7].

[0004] Among these genes, SCN5A, which encodes the α - subunit of the voltage - dependent sodium channel NaV1.5, is the major causative gene of Brugada syndrome [8]. However, the genetic etiology remains unknown in about 70 - 75% of cases, and a single mutation cannot fully explain the cause of the Brugada syndrome phenotype [9], so genetic testing is not sufficient as an independent diagnostic tool for this disease.

[0005] Histological changes in the right ventricular myocardium of type 1 Brugada syndrome patients demonstrate that Brugada syndrome is not caused solely by genetic factors [10,11], and the presence of inflammatory infiltrates and fibrosis in the right ventricular outflow tract has been reported in Brugada syndrome patients [12,13]. Furthermore, new evidence suggests a long-overlooked potential role of autoimmunity in Brugada syndrome in cardiac arrhythmias. The discovery of autoantibodies associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) has revealed the influence of autoimmunity in Brugada syndrome [14,15].

[0006] Autoantibodies can cause arrhythmias by interfering with ion channels and receptors involved in cardiac electrophysiology. For example, autoantibodies targeting β1-adrenergic receptors have been reported in various heart diseases, including ischemic cardiomyopathy and Chagas disease [16,17]. Furthermore, IgG antibodies against voltage-gated KCNQ1 K+ channels (Kv7.1 or KvLQT1) have been detected in patients with dilated cardiomyopathy, resulting in shortened QTc intervals

[18] . Autoantibodies targeting cardiac voltage-gated Na+ channels have also been found in patients with idiopathic severe AV block, leading to decreased sodium current (INa) density in rat cardiomyocytes

[19] .

[0007] Overall, to fully understand the disease, further investigation into the involvement of the immune system in Brugada syndrome, beyond genetic factors, is necessary.

[0008] Previous studies have shown abnormal protein distributions in Brugada syndrome cardiomyocytes, including α-actin, keratin-24, and connexin-43, which may affect the transport process of sodium channel complexes

[12] . These proteins have also been identified as potential targets for autoantibodies, suggesting an abnormal immune response. Therefore, the presence of NaV1.5 channel autoantibodies in Brugada syndrome plasma has not been definitively demonstrated to date.

[0009] Here, the inventors are addressing the pathophysiology of Brugada syndrome beyond genetic factors by investigating the presence and effects of autoantibodies against the NaV1.5 channel.

[0010] Specifically, the inventors developed an in vitro model of a NaV1.5 overexpression channel that mimics a physiological environment, exhibiting improved purification capabilities and the ability to test autoantibody binding under denatured and natural conditions. Previous studies have shown that autoantibodies against NaV1.5 can affect sodium ion currents in vivo in rats

[20] . This mechanism is consistent with the activity of autoantibodies against channels or receptor proteins seen in other diseases such as NMDAR encephalitis, where autoantibodies promote receptor internalization and induce electrophysiological changes in neurons [21-23].

[0011] By targeting autoimmunity, these groundbreaking findings hold promise for developing therapeutic strategies and improving patient care in Brugada syndrome. Mitigating the adverse effects of autoantibodies against NaV1.5 channels may lead to better management of Brugada syndrome and its associated arrhythmias. [Overview of the Initiative] [Problems that the invention aims to solve]

[0012] Therefore, the object of the present invention is an antibody against the NaV1.5 channel (SEQ ID NO: 1) and related isoforms for use as a biomarker for the diagnosis of Brugada syndrome in humans. [Means for solving the problem]

[0013] In fact, autoantibodies targeting the NaV1.5 channel (SEQ ID NO: 1) can cross-react with other NaV1.5 channel isoforms. Due to the high sequence similarity between the standard NaV1.5 channel and its isoforms, including the embryonic mutant nNaV1.5, autoantibodies initially produced against these alternative isoforms can also interact with cardiac NaV1.5 channels. Such cross-reactivity may contribute to the pathophysiology of Brugada syndrome by affecting the functional integrity of cardiac sodium channels and potentially exacerbating the risk of arrhythmias.

[0014] Accordingly, according to the present invention, the expression "NaV1.5 channel and associated isoforms" encompasses all NaV1.5 channels and all their variants, but is not limited to embryonic isoforms that share significant sequence homology with a standard NaV1.5 channel (SEQ ID NO: 1). The expression "significant sequence homology" refers to sequence homology greater than 85%, preferably greater than 90%, and more preferably greater than 95%, 96%, 97%, 98%, or 99%.

[0015] In a preferred embodiment, the antibody for use as a biomarker for the diagnosis of Brugada syndrome in humans according to the present invention targets the binding site in the extracellular loop of the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms.

[0016] However, in the presence of misfolded protein channels in Brugada patients, it cannot be ruled out that autoantibodies may target exposed binding sites on the inner channel protein.

[0017] Preferably, the antibody for use as a biomarker for the diagnosis of Brugada syndrome according to the present invention targets a binding site in the extracellular loop of the NaV1.5 channel and associated isoforms, wherein the binding site has the following sequence: i) VFALIGLQLFMGNLRHKCVRNFTALNGTNGSVEADGLVWESLDLYLSDPENYLLKNGTSDVLLCGNSSDAGTCPEGYRCLKAGENPDHGYTSFDSFAWAFLALFRL (Sequence ID 2) ii) FGKNYSELRDSDSGLLPRWHMMDFFHAFLIIFRILCG (Sequence ID 3) iii)SIMGVNLFAGKFGRCINQTEGDLPLNYTIVNNKSQCESLNLTGELYW TKVKVNFDNVGAGYLALLQ-1414 (Sequence ID 4) iv)VILSIVGTVLSDIIQKYFFSPTLFRVIRLARIGRIL(Sequence No. 5) v)IYSIFGMANFAYVKWEAGIDDMFNFQTFANSMLCLFQI(Sequence ID 6) vi)AGWDGLLSPILNTGPPYCDPTLPNSNGSRGDCGSPAVGILFFTT(Sequence ID 7) vii)SVEADGLVWESLDLYLSDPENYLLKNGTS(Sequence ID 8) viii)CLKAGENPDHGYTSFDSFAWAFLAL(Sequence ID 9) ix) DSDSGLLPRWHMMDFFHAFLII (Sequence ID 10) x)VNNKSQCESLNLTGEYWTKVK(Sequence ID 11) xi)GSGVILSIVGTVLSDIIQKYFFSPT(Sequence ID 12) xii)MANFAYVKWEAGIDDMFNFQTFANSMLCLF(Sequence ID 13) xiii)GWDGLLSPILNTGPPYCDPTLPNSNGSRGD(Sequence ID 14) or its fragment It can be selected from a group that includes [this].

[0018] The antibody targeting the NaV1.5 channel (SEQ ID NO: 1), preferably the antibody targeting the binding site in the extracellular loop of the NaV1.5 channel, more preferably the antibody targeting the binding site having a sequence selected from SEQ ID NOs: 2 to SEQ ID NO: 14, is an autoantibody that appears in a biological sample of a human suffering from Brugada syndrome.

[0019] The present invention further relates to an in vitro method for detecting the presence or detecting the amount of at least one of the antibodies of the present invention targeting the NaV1.5 channel in a biological sample of a human suspected of having Brugada syndrome. In a preferred embodiment of the present invention, the antibody targets the extracellular loop of the NaV1.5 channel of (SEQ ID NO: 1) and related isoforms.

[0020] In a preferred embodiment of the present invention, the human can be either asymptomatic or at high risk of Brugada syndrome due to family history, past events of atrial fibrillation and / or ventricular fibrillation, diabetes or obesity.

[0021] According to a preferred embodiment, the in vitro method comprises: a) contacting the biological sample with one or more antigens that specifically bind to an antibody against the NaV1.5 channel (SEQ ID NO: 1) and related isoforms; and b) detecting the binding of the antigen to the antibody in the biological sample and includes.

[0022] Preferably, the one or more antigens in step a) bind to an antibody targeting the extracellular loop of the NaV1.5 channel of (SEQ ID NO: 1) and related isoforms.

[0023] Preferably, the antigen is a fragment of 5 amino acids or more, more preferably 7 amino acids or more, derived from a binding site in the extracellular loop of the NaV1.5 channel. In a preferred embodiment, the binding site in the extracellular loop of the NaV1.5 channel is selected from the sequences of SEQ ID NOs: 2 to 14.

[0024] According to a preferred embodiment of the in vitro detection method for antibodies against NaV1.5 channels and associated isoforms of the present invention, the antigen is labeled.

[0025] In a more preferred embodiment of the in vitro method of the present invention, the biological sample is selected from the group consisting of plasma, peripheral blood mononuclear cells, whole blood, serum, peripheral blood, pericardial fluid, or a combination thereof. Preferably, the biological sample is plasma and / or peripheral blood mononuclear cells.

[0026] In a preferred embodiment of the in vitro detection method for antibodies against NaV1.5 channels and associated isoforms of the present invention, detection of antigen binding to the antibody is performed by ELISA, FACS analysis, or Western blotting.

[0027] A further object of the present invention is an antagonist for use as a therapeutic agent in the treatment of Brugada syndrome, which is an antagonist of the binding of the antibody to the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms. Preferably, the antibody targets the extracellular loop of the NaV1.5 channel (SEQ ID NO: 1).

[0028] For example, steroids such as prednisone, methylprednisolone, and dexamethasone, as well as drugs such as colchicine and hydroxychloroquine (Plaquenil), which can reduce the production of autoantibodies, are advantageous for treating Brugada syndrome.

[0029] The present invention further envisions a therapeutic agent specifically targeting antibodies against the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms for use in the treatment of Brugada syndrome. Preferably, the antibody targets the extracellular loop of the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms.

[0030] According to a preferred embodiment of the present invention, the therapeutic agent is characterized by comprising one or more antigenic fragments of the extracellular loop of the Nav1.5 channel (SEQ ID NO: 1) and associated isoforms.

[0031] Preferably, the autoimmune therapeutic agent comprises a sequence of about 10 amino acids or more derived from the extracellular loop of a NaV1.5 channel, and the extracellular loop is selected from the group of sequences SEQ ID NOs: 2 to 14.

[0032] Another possible therapeutic approach targets B cells, aiming to inhibit the production of autoantibodies that can cause immune complex-mediated inflammation. Drugs such as rituximab, ocryzumab, ofatumumab, and inebilizumab, which target B cells and disrupt the production of pathogenic autoantibodies, have already demonstrated efficacy in treating various B cell-mediated immune and autoimmune diseases and may be useful as therapeutic treatments.

[0033] Further therapeutic approaches for Brugada syndrome include the use of drugs that inhibit neonatal Fc receptors (FcRn), which affect the recycling and half-life of pathogenic autoantibodies. Nipocalimab, along with other FcRn inhibitors such as efgaltigimod, rozanolixizumab, and batoclimab, reduces levels of circulating autoantibodies by interfering with their recycling.

[0034] This approach can reduce autoantibody load and mitigate their pathological effects on NaV1.5 channels. By targeting the FcRn-mediated recycling pathway, these drugs reduce autoantibody titers without broadly suppressing the immune system, thus offering a safer alternative to conventional immunosuppressive therapy.

[0035] Alternatively, nanobody-based polyvalent or multispecific formulations targeting Ab-Nav1.5 are intended to restore normal activity of the NaV1.5 channel. Furthermore, these nanobodies serve as useful tools for in vivo imaging of cardiac substrates affected by Brugada syndrome, enabling effective monitoring.

[0036] The above treatment strategy is based on a significant decrease in sodium current observed in the presence of autoantibodies in a mouse model, followed by normalization upon their removal (see data shown in Figures 9 to 10).

[0037] The present invention further envisions the use of antagonists or autoimmune therapies in combination with other treatments for Brugada syndrome to achieve a more comprehensive and effective treatment.

[0038] The present invention further relates to a kit for detecting antibodies against a NaV1.5 channel (SEQ ID NO: 1) and associated isoforms in a biological sample, comprising one or more antigens that specifically bind to an autoantibody against the NaV1.5 channel. Preferably, the one or more antigens specifically bind to an antibody targeting the extracellular loop of the NaV1.5 channel (SEQ ID NO: 1). Even more preferably, the antigens comprise one or more antigenic fragments or epitopes belonging to a binding site in the extracellular loop of the NaV1.5 protein, wherein the binding site in the extracellular loop is selected from the sequence group of SEQ ID NOs: 2 to 14.

[0039] In preferred embodiments of the kit of the present invention, the antigen is labeled or conjugated to a solid support. Preferably, the antibody is fluorescently labeled.

[0040] The solid support is preferably a multi-well plate. Preferably, the kit is an ELISA kit.

[0041] The present invention further relates to an antigenic fragment or epitope belonging to a binding site in the extracellular loop of a NaV1.5 channel, selected from the group consisting of sequences SEQ ID NOs: 2 to 14.

[0042] Furthermore, the present invention relates to the use of one or more autoantibodies against the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms for the diagnosis of Brugada syndrome in humans, by the (qualitative or quantitative) detection of their presence in a biological sample, wherein the biological sample is selected from the group consisting of plasma, human peripheral blood mononuclear cells, whole blood, serum and peripheral blood, pericardial fluid, or a combination thereof. According to a preferred embodiment of the present invention, the detection step is carried out by using one or more antigens that specifically bind to the autoantibodies against the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms. In a more preferred embodiment of the present invention, the one or more antigens are selected from antigenic fragments or epitopes belonging to binding sites in the extracellular loop of the NaV1.5 channel, consisting of sequences SEQ ID NOs: 2 to 14.

[0043] Furthermore, the present invention intends to use one or more autoantibodies of the present invention as markers for monitoring the effectiveness of therapeutic treatment for Brugada syndrome disease by detecting autoantibody titers in biological samples of treated patients. The biological sample may be selected from the group consisting of plasma, human peripheral blood mononuclear cells, whole blood, serum, peripheral blood, peripheral fluid, or a combination thereof. According to a preferred embodiment, the therapeutic treatment may be a pharmacological treatment or an interventional surgical treatment, such as ablation.

[0044] The present invention further relates to an animal model obtained by injecting plasma derived from a patient suffering from Brugada syndrome containing autoantibodies against the above-mentioned NaV1.5 channel (SEQ ID NO: 1) and / or related isoforms.

[0045] Alternatively, the present invention relates to an animal model obtained by injecting one or more antigens that specifically bind to antibodies against the above-mentioned NaV1.5 channel (SEQ ID NO: 1) and associated isoforms, wherein the antigen is an antigenic fragment or fragment thereof selected from the group of sequences SEQ ID NOs: 2 to 14.

[0046] In a preferred embodiment of the present invention, the animal model is a rodent, preferably a mouse.

[0047] In a preferred embodiment, the animal model is a humanized animal model.

[0048] The "humanized animal model" according to the present invention is an animal, typically a rodent such as a mouse, that has been modified to express human genes, proteins, or cells. This allows the animal to more closely mimic human physiological and pathological processes. In this case, the humanized animal model is achieved by injecting antigens that target specific antibodies, and helps in the study of NaV1.5 channels and their associated isoforms in an environment that better represents human biology.

[0049] The present invention also envisions a method for identifying compounds for the prevention and / or treatment of Brugada syndrome, comprising the steps of administering a candidate compound to the above-described animal model and detecting changes in antibodies against the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms in the animal model compared to a control. Hereinafter, the present invention will be described, for non-limiting illustrative purposes, according to their preferred embodiments, particularly with reference to the accompanying figures. [Brief explanation of the drawing]

[0050] [Figure 1] Figure 1 shows the detection of autoantibodies against the NaV1.5 channel in the plasma of patients with Brugada syndrome. Panel A is a Western blot: proteins from lysed cells were separated by one-dimensional SDS-PAGE. The NaV1.5 protein band was observed at 250 kd in lysates of transfected HEK cells (HEK-NaV1.5) but not in lysates of untransfected (HEK-WT) cells. Co-localization of IgG from BrS patients with and without the SCN5A mutation was detected with anti-rabbit anti-NaV1.5 and anti-human IgG, but not in healthy controls. Panel B shows immunoprecipitation (IP) of NaV1.5 protein with purified IgG from the plasma of BrS patients and healthy controls (n=3). [Figure 2] Figure 2 shows a Western blot analysis demonstrating that IgG derived from Brugada syndrome plasma can specifically bind to NaV1.5 derived from protein lysate of mouse cardiac tissue. [Figure 3] Figure 3 shows representative fluorescence images verifying the presence of NaV1.5 autoantibodies in plasma from patients with Brugada syndrome. Panel A) Representative fluorescence images showing BrS-positive and BrS-negative samples. Red fluorescence highlights NaV1.5 expression, while green fluorescence indicates the presence of bound human antibodies. The same plasma was tested with both transfected and untransfected cells (HEK-WT). Panel B) Representative fluorescence images showing BrS-positive and BrS-negative plasma. Red fluorescence indicates NaV1.5 expression, while green fluorescence functions as an indicator of human IgG. [Figure 4] Figure 4 shows Western blot analysis of cell lysates derived from NaV1.5-transfected HEK293A cells treated with anti-human IgG antibody and then pre-stained with anti-NaV1.5 antibody (Panel A), as well as lysates incubated with boiled BrS patient plasma before exposure (Panel B). [Figure 5]Figure 5 shows the detection of autoantibodies against NaV1.5 protein from plasma-derived patients with Brugada syndrome at the time of diagnosis (right panel) and after catheter ablation (left panel). Western blot analysis: Proteins from lysed cells were separated using one-dimensional SDS-PAGE. The NaV1.5 protein band at 250 kd was observed in lysates from transfected HEK cells (HEK-NaV1.5) but not in lysates from untransfected cells (HEK-WT). Examination using anti-rabbit anti-NaV1.5 and anti-human IgG antibodies revealed that IgG co-localized in lysates from Brugada syndrome patients with and without the SCN5A mutation, but not in lysates from patients after ablation (PA). [Figure 6] Figure 6 shows the detection of autoantibodies against the NaV1.5 protein in the plasma of patients with Brugada syndrome. Western blot analysis: Proteins from lysed cells were separated by one-dimensional SDS-PAGE. The NaV1.5 protein band at 250 kd was observed in lysates from transfected HEK cells (HEK-NaV1.5), but not in lysates from untransfected cells (HEK-WT). Examination using anti-rabbit anti-NaV1.5 and anti-human IgG antibodies revealed that IgG co-localized in lysates from BrS patients with and without the SCN5A mutation, but not in lysates from healthy controls. [Figure 7] Figure 7 shows a comparison of representative fluorescence images of Brugada syndrome-positive and Brugada syndrome-negative plasma. Panel A: Representative fluorescence images showing BrS-positive and BrS-negative plasma. Red fluorescence indicates NaV1.5 expression, while green fluorescence functions as an indicator of human IgG. Panel B: Manders coefficient. [Figure 8]Figure 8 shows the effect of autoantibodies derived from patients with Brugada syndrome on sodium current. Panel A) Sodium current profiles in HEK293A cells overexpressing NaV1.5, incubated with either a control (CTR, left panel) or serum from a Brugada syndrome patient (BrS, right panel, filled circles). Panels B-C) ​​Mean current-voltage relationships (I-V) (N=6, n=70, open circles for control; N=8, n=91, filled circles for BrS serum). [Figure 9] Figure 9 shows the effect of BrS patient plasma on inward current in hiPSC-derived cardiomyocytes (hiPSC-CMs). Panel A) shows current traces (families) of TTX-sensitive sodium current induced by a voltage step protocol after 1 hour incubation with untreated (left) or BrS patient plasma (right), showing an approximately 50% reduction in current density compared to untreated cardiomyocytes. Panels B-C show the mean current-voltage relationship and voltage dependence of channel activation of TTX-sensitive sodium current obtained from untreated cells (black triangles, n=26, N=3 independent differentiations) or cells incubated with BrS patient plasma (white circles, n=33, N=3 plasmas) (*p<0.5). Panel D shows traces of nifedipine-sensitive calcium current in untreated (left) or BrS patient plasma-incubated (right) hiPSC-derived cardiomyocytes, showing that plasma incubation does not affect current density. Panels E-F show the IV curves and voltage dependence of activation of nifedipine-sensitive calcium current in untreated cells (black triangles, n=22, N=3 independent differentiations) and cells incubated with BrS patient plasma (n=30, N=3 BrS plasma), respectively. [Figure 10]Figure 10 shows the electrocardiogram response to plasma in vivo. Panels A and B show ECG recordings of mice before (Panel A) and after (Panel B) administration of plasma derived from BrS patients. After administration, the ECGs showed a Brugada-like ECG pattern commonly observed in humans in the bipolar leads, specifically ST elevation in lead III and mirror-image ST depression in leads I and II. Panels C and D show the ECG results of mice treated with plasma from non-BrS control subjects. In particular, no changes in the ECG were observed after infusion (Panel D), indicating the absence of arrhythmic activity. Panels E and F show ECG traces of mice administered antibody-depleted BrS plasma. No ECG abnormalities were observed after infusion. The ECG recordings show three bipolar lead configurations (leads I, II, and III, respectively). [Figure 11] Figure 11 shows the identification of putative autoantibody binding sites on the NaV1.5 protein. Panel A) Amino acid sequence of the NaV1.5 channel (SEQ ID NO: 1); Panel B) Identification of six potential regions that act as autoantibody binding sites. Ten amino acids important for binding on a solid support are highlighted in yellow. Panel C) PyMol structure showing the core and extracellular loop of the NaV1.5 channel. [Figure 12-1] Figure 12 shows a scan of peptide microarrays of human plasma from 20 patients with Brugada syndrome (Br1200, Dg2675, Dg1936, Br1211, Dg2821, Dg2533, Dg2429, Dg2388, Dg2830, Dg2655, Dg2677, Dg2828, Br1196, Dg2720, Dg2818, Dg2819, Br1145, Br938, Br1204, Br1215) that were analyzed. [Figure 12-2] Same as above. [Modes for carrying out the invention]

[0051] The following embodiments are merely illustrative and should not be considered to limit the scope of the present invention. [Examples]

[0052] (Example 1): Detection of NaV1.5 channel autoantibodies in plasma from Brugada patients method (Human sample) This study included Brugada syndrome (BrS) patients diagnosed at the arrhythmia department of IRCCS Policlinico San Donato

[24] . Participants were classified into three main subgroups: 1) BrS patients with SCN5A mutations; 2) BrS patients without SCN5A mutations; and 3) healthy controls. In addition, five patients underwent catheter ablation. The study procedure was approved by the local institutional ethics committee, and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki (NCT02641431;NCT03106701) [25,26]. All patients met the diagnostic criteria for BrS, including the presence of spontaneous or drug-induced type 1 Brugada ECG patterns. Clinical data, medical history, 12-lead ECG recordings, and implantable cardioverter-defibrillator (ICD) results were collected from medical records.

[0053] (Cell culture) HEK293 cells were cultured at 37°C under 5% CO2 in DMEM high-glucose medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% glutamine.

[0054] (Mouse model) Adult C57BL-6 mice were housed under standard conditions including a 12-hour light / dark cycle and free access to food and water. The environment in which the mice were kept was strictly controlled.

[0055] (Whole exome sequencing) All patients were examined using a next-generation sequencing panel of genes containing SCN5A from peripheral blood extracted DNA. DNA was extracted from peripheral blood and processed to obtain a library containing approximately 575 kb of genomic DNA using a 50 nanogram DNA input. The library was subjected to deep sequencing, deduplication, and filtering of low-quality reads to achieve an average target coverage of 100X. Next-generation sequencing data were validated using Sanger sequencing according to the guidelines of the American College of Medical Genetics (ACMG)

[27] . Variants were annotated using information from well-known public databases, including dbSNP, dbNSFP, ExAC, and ClinVar.

[0056] (Plasma collection and processing) Blood (25 ml) was centrifuged at 1000 g for 15 minutes to isolate the plasma, and then 2000 g of the supernatant was centrifuged for 15 minutes. The supernatant was collected, divided into aliquots, and stored at -20°C.

[0057] (IgG isolation) IgG antibodies were isolated using the PureProteome® Protein G magnetic bead system. Isolation was performed under non-denaturing conditions of high salinity and near-neutral pH.

[0058] (Preparation of stable cell lines and transient transfection) To express the NaV1.5 channel protein, full-length cDNA encoding human SCN5A was synthesized and cloned into pcDNA3.1(+). HEK293 cells were transfected with the pcDNA3.1(+) / SCN5A construct using ViaFect transfection reagent (Promega) according to the manufacturer's instructions. Cells were grown in a medium consisting of Dulbecco's modified Eagle medium (DMEM, Life Technologies) selected with a specific concentration of G418, supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM glutamine (Merck), and 1X penicillin / streptomycin (Euroclone). Cells were maintained at 37°C in a 5% CO2 and 95% humidified air atmosphere. The harvested cells were lysed, and the clarified lysate was collected after centrifugation. Western blot analysis confirmed the successful transfection and expression of the NaV1.5 channel.

[0059] (Western blot) Cells were lysed in a cell lysate containing 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS, supplemented with protease and protein phosphatase inhibitors. Total protein concentration was measured using a BCA assay. Proteins were denatured and reduced in a Laemmli-β-mercaptoethanol mixture at 100°C for 5 minutes. Subsequently, 30 μg of protein was loaded onto a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. After blocking nonspecific binding, the membrane was incubated overnight at 4°C with primary rabbit monoclonal anti-NaV1.5 antibody (1:2000 dilution, clone D9J7S, Cell Signalling). After washing, the membrane was incubated at room temperature for 1 hour with a suitable anti-rabbit-AlexaFluor546 labeled secondary antibody (1:2000 dilution). After further washing, the membranes were incubated with patient or control plasma diluted 1:3 in PBS at room temperature for 2 hours. After further washing, a secondary antibody against human IgG-FITC was added at room temperature for 1 hour. Immunoreactivity bands were visualized using an enhanced chemiluminescence detection kit (ECL Advance, GE Healthcare).

[0060] (NaV1.5 immunoblot) HEK293A cells overexpressing NaV1.5 were lysed using RIPA buffer containing a cocktail of protease and phosphatase inhibitors, then centrifuged at 15,000 rpm at 4°C for 10 minutes, and the supernatant was collected. Protein concentration was determined by BCA assay (Pierce). The proteins were denatured and reduced with Laemmli buffer containing β-mercaptoethanol (Bio-Rad), loaded onto a 10% SDS-PAGE gel (Protean Tgx Stain-Free, Bio-Rad) for electrophoresis, and then transferred to a nitrocellulose membrane. The membrane was blocked, probed overnight at 4°C with primary anti-NaV1.5 antibody (1:2000 dilution, Cell Signaling, clone D9J7S), washed, and incubated with secondary anti-rabbit IRDye 800 CW antibody (1:2000 dilution, LI-COR Biosciences). Next, the membranes were incubated with plasma from BrS patients, either boiled at 100°C for 10 minutes or left untreated, and then stained with a secondary anti-human IgG-HRP antibody (1:2000 dilution, Bio-Rad). The bands were visualized using the ECL Advance kit (GE Healthcare) and imaged using the ChemiDoc MP system (Bio-Rad).

[0061] (Immunoprecipitation of NaV1.5 channels from protein lysates of mouse heart tissue) To collect cardiac samples, animals were anesthetized by intraperitoneal injection of 0.5 mg / kg medetomidine (Orion Pharma Srl) and 100 mg / kg ketamine (Merial), both diluted in physiological saline. Once the animals were completely unconscious, the thoracic cavity was opened, and the heart was perfused with 1 ml of 1M KCl to induce diastolic arrest. 0.9% physiological saline was then flowed through a cannula inserted into the left ventricle. The heart was then removed, and the left ventricle was separated from the atria and right ventricle. The left ventricle was then dissected into 3 mm thick sections using a specific stainless steel cardiac matrix (Roboz Surgical Instruments) to prepare apical, midline, and basal specimens. The apical section was incubated with 500 ml of RIPA cell lysate and homogenized using a tissue homogenizer, Lyser® (Qiagen). Each sample was subjected to three homogenization cycles, each lasting 5 minutes at an oscillation rate of 25 times per second. The homogenates were kept on ice for 30 minutes, then centrifuged at 10,000 rcf for 10 minutes at 4°C. After centrifugation, the supernatant of each tissue sample was transferred to a new tube, and the total protein content was quantified using the BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0062] 300 μg of total protein was incubated with Dynabeads® Protein G conjugated with IgG from BrS plasma or a control, and the eluted fraction was subjected to Western blot analysis stained with NaV 1.5 as described above.

[0063] (Immunoprecipitation of NaV1.5 derived from transfected HEK-293 cells) Immunoprecipitation of NaV1.5 channel proteins from transfected HEK-293 cells was performed using patient and healthy control serum IgG specific to the NaV1.5 channel. Immunoprecipitation was performed by incubating the antibody with Dynabeads Protein G at room temperature for 30 minutes. After washing, the beads were incubated with whole cell lysates at room temperature for 2 hours with orbital shaking. After further washing, Laemmli-β-mercaptoethanol sample buffer was added to the beads, and the eluted fraction was subjected to SDS-PAGE.

[0064] (Immunofluorescence in mouse tissue) Immunofluorescence assays were performed on adult C57BL-6 mouse cardiac tissue. 12 μm thick left ventricular sections were prepared using a cryostat and placed on gelatin-coated tissue slides. The sections were thawed, rehydrated, and subjected to antigen unmasking. Nonspecific binding was blocked using a solution containing normal donkey serum and bovine serum albumin. After incubation with patient plasma diluted 1:50 in PBS containing 2% NDS and 2% BSA, the sections were washed and incubated with an anti-human IgG-FITC secondary antibody. Subsequently, the sections were incubated with primary rabbit monoclonal anti-NaV 1.5 antibody (1:200 dilution; clone D9J7S, Cell Signaling), washed, and incubated with a suitable anti-rabbit secondary antibody labeled with Cy3. After further washing, the sections were mounted using Vectashield mounting medium containing DAPI. Images were acquired using a Leica Thunder microscope (×40 objective lens).

[0065] (Cell-based assay immunofluorescence (CBA-IF)) To evaluate the binding of IgG-NaV1.5 autoantibodies, live HEK293 WT cells and HEK293 cells expressing NaV1.5 protein were used. Cells were plated onto coverslips and incubated with patient plasma diluted 1:3 in DMEM HG supplemented with 1% L-glutamine, 1% penicillin-streptomycin, and 5% thermo-inactivated serum. After washing, cells were fixed in cold methanol and subjected to blocking. After incubation with NaV1.5 antibody, cells were washed and stained with rabbit secondary antibody and FITC anti-human IgG. Nuclear staining was performed using DAPI. Images were acquired using a Leica Thunder microscope (×40 objective lens).

[0066] (Quantification of colocalization) Confocal microscope images were randomly acquired for all groups, and colocalization analysis was performed using the JACoP plugin of FIJI software. Manders coefficients were calculated to quantify colocalization.

[0067] (Cellular electrophysiology) To record sodium currents from HEK293A cells transiently transfected with NaV1.5 channels, a chip-based automated planar patch clamp system, Patchliner (Nanion Technologies GmbH, Munich, Germany), was used. After incubation at 37°C for 1 hour in 5% CO2 with 5% BRS or control-derived serum, the cells were mildly trypsinized and resuspended in an extracellular low-sodium recording solution from Nanion (reference 08-3004, ionic composition in mM units: 80 NaCl, 60 NMDG, 4 KCl, 2 CaCl2, 1 MgCl2, 5 D-glucose monohydrate, 10 Hepes; adjusted to pH 7.4 with HCl, 289 mOsm). Untreated cells were used as an internal reference. The cells were then incubated at 4°C for 20 minutes to improve membrane stability. All recordings were performed at room temperature with the entire cell composition using a medium-resistance NPC-16 chip. At least two chips were used for each condition on each experimental day. CsF-based intracellular (reference 08-3008, ionic composition in mM: 10 EGTA, 10 Hepes, 10 CsCl, 10 NaCl, 110 CsF; pH adjusted to 7.2 with CsOH, 280 mOsm) and extracellular seal enhancer (reference 08-3011, ionic composition in mM: 80 NaCl, 60 NMDG, 4 KCl, 10 CaCl2, 1 MgCl2, 5 D-glucose monohydrate, 10 Hepes; pH adjusted to 7.4 with HCl, 313 mOsm) solutions were also provided by Nanion. To reduce any bias due to transfection variability, at least one BrS serum and one control serum from a healthy donor were tested on each individual experimental day. Cell capacitance and series resistance were automatically corrected by Patchliner. All currents were sampled at 50 kHz. The current-voltage (IV) relationship and the voltage dependence of sodium current activation were obtained by applying a protocol with 50 ms increments in the range of -80 to +60 mV (holding potential -120 mV).Raw traces recorded by the HEK293A amplifier were exported using a custom-built Python tool, and individual traces were analyzed using Clampfit 10.7 (Molecular Devices, San Jose, California, USA), Origin Pro (OriginLab, Norhampton, Massachusetts, USA), and GraphPad Prism (GraphPad Software, Boston, Massachusetts, USA). Current density was calculated by dividing the current amplitude (pA) by the cell capacitance (pF) of each cell.

[0068] (Current measured by hiPSC-CM) An inward current was induced in the whole cell composition by manual patch clamp at 37°C using a step protocol with a voltage of -80 to 60 mV and a duration of 150 ms (maintaining -80 mV). This protocol was applied in the absence of any drug, in the presence of 10 μM nifedipine, and in the presence of 10 μM nifedipine and 30 μM TTX. Nifedipine-sensitive ICaL and TTX-sensitive INa were obtained during analysis, followed by subtraction. The intracellular solution was (mM) 135 CsCl, 10 NaCl, 5 EGTA, 2 CaCl2, 2 TEA-Cl, 10 HEPES, 2 MgATP, with CsOH at pH 7.2. The extracellular solution contained 80 mM NaCl.

[0069] (Animal and electrocardiogram examinations) The procedures involving mice were carried out in accordance with the animal protocol guidelines described by the Institutional Animal Care and Use Committee (IACUC) certification number 425 / 2022 / PR of the San Raffaele Scientific Institute (Milan, Italy). 50-week-old mice C57BL-6 were kept at room temperature with ample access to water and standard solid feed on a 12-hour light / dark schedule. Both mice were anesthetized by intraperitoneal injection of 0.5 mg / kg medetomidine (Orion Pharma Srl) and 100 mg / kg ketamine (Merial), both diluted in saline. Body weight was determined before each study, and body temperature was continuously monitored and maintained at 37 ± 0.5°C using a constant-temperature blanket system equipped with a rectal thermometer probe (Harvard Apparatus, Holliston, Massachusetts, USA). Prior to intravenous injection into anesthetized mice, 200 μl of plasma derived from BrS (n=4) and CTR (n=3) was preheated at 56°C for 20 minutes.

[0070] In short, ECGs were performed continuously from 10 minutes after induction of anesthesia to 30 minutes after plasma injection using four subcutaneous needle electrodes (stainless steel, 27 gauge, 12 mm in length; SEI EMG srl, Cittadella, Italy). Two needles were inserted into the forelimb, one needle electrode into the left hindlimb, and the other needle electrode was placed in the right hindlimb as ground. A consistent induction configuration was maintained, with no changes in the polarity or placement of the subcutaneous needle electrodes before, during, or after plasma administration. Specifically, the leads were placed in a standard Eindhoven configuration for mice to ensure that each mouse followed the same procedure for accurate results. The induction configuration remained unchanged throughout the plasma infusion study. The following is a general description of the lead placement for mouse ECGs.

[0071] - Lead I: This lead measures the potential difference between the left and right forelimbs (or arms). Electrodes are placed on both the right forelimb (negative electrode) and the left forelimb (positive electrode). This is shown in the first line of every ECG experiment. - Lead II: This lead measures the potential difference between the right forelimb (negative electrode) and the left hindlimb (positive electrode). The electrodes are placed on the right forelimb and left hindlimb. This is shown in the second line of all ECG experiments. - Lead III: This lead measures the potential difference between the left forelimb (negative electrode) and the left hindlimb (positive electrode). The electrodes are placed on the left forelimb and left hindlimb. This is shown on the third line of all ECG experiments. - Grounding electrode: To stabilize the signal and reduce noise, the grounding electrode is placed in the neutral region, on the right hind limb.

[0072] After connecting the needle electrode to an amplifier (Micromed, Mogliano Veneto, Italy) via a flexible cable, the ECG signal was recorded using System-Plus software (Micromed, Mogliano Veneto, Italy) and sampled at 256 Hz (16-bit) using a bandpass filter between 1 and 70 Hz. All animal experiments were performed without prior knowledge of the origin of the plasma samples to ensure the completeness of the results.

[0073] result (Detection of autoantibodies against NaV1.5 protein in Brugada syndrome plasma) A cohort of 100 subjects was included in the study. Western blot analysis was performed first to detect the presence of IgG autoantibodies against the NaV1.5 channel in patient-derived plasma samples. Of the BrS patients who tested positive for Ajmaline (n=53), 48 patients without the SCN5A mutation and 5 patients with the SCN5A variant showed autoantibodies. In contrast, plasma samples from 47 subjects who tested negative for Ajmaline did not show the presence of autoantibodies (Figure 1A and Figure 6). To further confirm the presence of IgG against the NaV1.5 channel, an immunoprecipitation assay was performed. IgG isolated from the plasma of patients positive for autoantibodies showed binding to the NaV1.5 protein extracted from HEK cell lysates (Figure 1B).

[0074] (Immunoprecipitation of mouse NaV1.5 using BrS anti-NaV1.5 autoantibody) Western blot analysis showed that IgG derived from BrS plasma can specifically bind to NaV1.5 derived from protein lysate of mouse cardiac tissue (Figure 2).

[0075] Magnetic beads coated with IgG derived from BrS patients were incubated with mouse ventricular protein extracts. The immunoprecipitation (IP) and immunodepletion (I-) fractions were then separated by Western blotting (left blot).

[0076] As revealed by staining with a commercially available anti-NaV1.5 antibody (right-hand blot), the presence of NaV1.5 protein in the IP fraction confirmed the presence of anti-NaV1.5 IgG in BrS plasma.

[0077] (Binding of NaV1.5 to cells overexpressing the NaV1.5 channel by plasma IgG from BRS patients) To investigate the binding of NaV1.5 by plasma IgG from BrS patients, dual immunofluorescence labeling was performed on mouse cardiac slides using an anti-NaV1.5 monoclonal antibody and plasma from BrS patients and healthy controls. Co-localization of immunofluorescence signals was observed when using the monoclonal anti-NaV1.5 antibody and plasma from BrS patients, but no signal was observed in healthy control plasma (Figure 3A). Similar results were obtained by immunolabeling cells overexpressing the NaV1.5 channel using plasma from BrS patients. The anti-NaV1.5 antibody from BrS patients specifically bound to NaV1.5-transfected cells but not to mock-transfected cells (Figures 3B and 7).

[0078] Furthermore, Western blot analysis of cell lysates derived from BrS patient plasma and NaV1.5-transfected HEK293A cells treated with anti-human IgG antibody, after pre-staining with anti-NaV1.5 antibody, supports the specific interaction between BrS patient autoantibodies and NaV1.5 (Figure 4, Panel A).

[0079] The same experiment, performed on lysates incubated with boiled BrS patient plasma before exposure, showed a loss of specific anti-NaV1.5 IgG binding to NaV1.5 protein, suggesting that thermal denaturation of IgG causes the loss of their binding ability (Figure 4, Panel B).

[0080] (Undetectable autoantibodies against NaV1.5 in BRS patients after catheter ablation) To evaluate the impact of catheter ablation on the presence of autoantibodies against NaV1.5, a subset of five patients from the initial cohort was analyzed 6 months after the ablation procedure. Western blot analysis showed that these patients did not have IgG autoantibodies against NaV1.5 after epicardial ablation (Figure 5).

[0081] (Effect of autoantibodies derived from Brugada syndrome patients on sodium current) The authors of this invention conducted in vitro experiments to evaluate the effect of IgG removal on sodium current in HEK293 cells and hiPSC-derived cardiomyocytes (see the following paragraph).

[0082] In particular, Figure 8 shows that autoantibodies derived from patients with Brugada syndrome reduce sodium current. To record sodium current in HEK293 cells overexpressing the NaV1.5 channel, automated patch clamping was used with the whole cell composition and incubated for 1 hour with either 5% BrS patient plasma or plasma from a healthy donor.

[0083] The results showed that the mean peak current density at -20mV was measured at -211.2±21pA / pF (n=79) under the latter condition, compared to -122.3±12pA / pF (n=100) under the former condition, indicating a significant decrease of approximately 40% in current density. The current density recorded in untreated HEK293 cells overexpressing NaV1.5 as an internal reference was 169.8±12 (n=54), which was no different from the current density obtained in cells incubated with plasma from healthy donors. The data demonstrate a significant decrease (approximately 40%) in inward sodium current due to plasma exposure from Brugada syndrome compared to control plasma.

[0084] (Effect of Brugada syndrome patient plasma on the inward current of hiPSC-derived cardiomyocytes (hiPSC-CM)) Panels A-F of Figure 9 show the effect of plasma from Brugada syndrome patients on inward current in hiPSC-CM. These results strongly suggest the specificity of the effect of BrS plasma on sodium current.

[0085] (Effects of plasma injection from BrS patients in wild-type mice) To further demonstrate the functional role of autoantibodies against NaV1.5 channels in the pathophysiology of Brugada syndrome, the authors performed in vivo experiments in adult C57BL-6 mice to evaluate the effect of IgG on sodium current.

[0086] The electrophysiological characteristics of mice were evaluated using ECG. Seven mice expressing the wild-type isoform of the NaV1.5 channel were administered plasma intravenously from four BrS patients and control subjects under general anesthesia, and continuous ECG monitoring was performed. Mice administered with BrS plasma developed Brugada ECG ST segment abnormalities, followed by compound malignant arrhythmias (ventricular arrhythmias and atrioventricular [AV] block), leading to cardiac arrest and death (Figures 10A-10B).

[0087] However, mice administered with control plasma showed no ST elevation or conduction disorders, and no ECG changes (Figures 10C-10D). Furthermore, removal of autoantibodies from the plasma before injection prevented the Brugada-like phenotype in mice (Figures 10E-10F). Previously, administration of the same BrS plasma containing autoantibodies resulted in a coved-type ST elevation pattern and a malignant arrhythmia phenotype (Figures 10A-10B). This evidence supports the idea that therapeutic approaches and / or agents that can reduce autoantibody levels according to the present invention can reduce the risk of autoantibody-induced arrhythmias.

[0088] (Example 2): Identification of putative NaV1.5 autoantibody binding sites on NaV1.5 channel proteins The human NaV1.5 channel has the following amino acid sequence.

[0089] [ka]

[0090] (Prediction of NaV1.5 autoantibody binding sites on NaV1.5 channel proteins) To explore binding sites on NaV1.5 channel proteins for autoantibodies, molecular modeling was performed using crystal structures of NaV1.5 channel proteins downloaded from Uniprot SCN5A (Figure 11). Pymol software was used to predict favorable interactions between autoantibodies and target proteins, enabling rational design of binding sites.

[0091] (Epitope discovery) To avoid cleavage of peptides, the sequences of loops 1-6 of NaV1.5 (DI S5-S6(263-368), DII S5-S6(861-897), DIII S5-S6(1349-1414), DIV S3-S4(1598-1634), DIV S5-S6(1670-1707)&DIV S5-S6(1711-1754)) were extended using neutral GSGGSSG linkers (SEQ ID NO: 15) at the N-terminus and C-terminus. The extended sequences were converted into 15-amino acid peptides with 14-amino acid peptide duplication. The resulting NaV1.5 peptide microarray had 327 different peptides printed in overlapping layers (654 peptide spots), and was further surrounded by additional control peptides for HA (YPYDVPDYAG SEQ ID NO. 16, 40 spots) and polio (KEVPALTAVETGAT SEQ ID NO. 17, 38 spots).

[0092] After subjecting the microarrays to Rockland's blocking buffer MB-070 for 30 minutes, they were pre-stained with the secondary antibody goat anti-human IgG(Fc) DyLight680 (0.1 μg / ml) in incubation buffer at room temperature for 45 minutes to investigate background interactions that could interfere with the main assay.

[0093] Furthermore, NaV1.5 peptide microarrays were diluted 1:150 in the plasma of 20 Brugada syndrome patients (Br1200, Dg2675, Dg1936, Br1211, Dg2821, Dg2533, Dg2429, Dg2388, Dg2830, Dg2655, Dg2677, Dg2828, Br1196, Dg2720, Dg2818, Dg2819, Br1145, Br938, Br1204, Br1215) and incubation buffer (PBS, pH 7.4, 0.05% Tween 20; washed 3 × 10 seconds with 10% Rockland Blocking Buffer MB-070), incubated at 4°C for 16 hours under orbital shaking at 140 rpm, and then stained with secondary antibodies.

[0094] Readings were performed using an Innopsys InnoScan 710-IR microarray scanner. Additional HA peptides surrounding the peptide microarray were simultaneously stained with mouse monoclonal anti-HA(12CA5) DyLight800 (0.2 μg / ml) at room temperature (PBS, pH 7.4, 0.05% Tween 20; washed 3 × 10 seconds with 10% Rockland blocking buffer MB-070) for 45 minutes.

[0095] Spot intensity quantification and peptide annotation were performed based on 16-bit grayscale TIFF files, which have a higher dynamic range than the 24-bit colorized TIFF files shown in this report. Microarray image analysis was performed using PepSlide® Analyzer, a software algorithm that decomposes the fluorescence intensity of each spot into raw data, foreground, and background signals, and calculates the average of the median foreground intensity. An intensity map was generated based on the averaged foreground median intensity, and interactions in the peptide map were highlighted with an intensity color code, indicating strong interactions in red and weak interactions in white, depending on the spot intensity. An Innopsys InnoScan 710-IR microarray scanner was used, with a resolution of 20 μm and scan gains set to 50 at low laser power (680 nm, red) and 10 at high laser power (800 nm, green).

[0096] Using molecular modeling, the authors identified six putative binding sites (DI-DVI) for autoantibodies on the extracellular loop of the NaV1.5 channel protein (bold in SEQ ID NO: 1), as shown in Figure 11.

[0097] DI S5-S6(263-368)106 amino acids: VFALIGLQLFMGNLRHKCVRNFTALNGTNGSVEADGLVWESLDLYLSDPENYLLKNGTSDVLLCGNSSDAGTCPEGYRCLKAGENPDHGYTSFDSFAWAFLALFRL(Sequence ID 2) DII S5-S6 (861-897) 37 amino acids: FGKNYSELRDSDSGLLPRWHMMDFFHAFLIIFRILCG(Sequence ID 3) DIII S5-S6 (1349-1414) 66 amino acids SIMGVNLFAGKFGRCINQTEGDLPLNYTIVNNKSQCESLNLTGELYWTKVKVNFDNVGAGYLALLQ(Sequence ID 4) DIV S3-S4 (1598-1634) 36 amino acids VILSIVGTVLSDIIQKYFFSPTLFRVIRLARIGRIL (Sequence No. 5) DV S5-S6 (1670-1707) 38 amino acids IYSIFGMANFAYVKWEAGIDDMFNFQTFANSMLCLFQI(Sequence ID 6) DVI S5-S6 (1711-1754) 44 amino acids AGWDGLLSPILNTGPPYCDPTLPNSNGSRGDCGSPAVGILFFTT(Sequence ID 7)

[0098] (Epitope identification in human plasma of Brugada patients) NaV1.5 peptide microarrays were incubated with human plasma containing Dg2677, Dg2818, Br1200, Dg2675, Dg1936, Br1211, Dg2821, Dg2533, Dg2429, Dg2388, Dg2830, Dg2655, Dg2828, Br1196, Dg2720, Dg2819, Br1145, Br938, Br1204, and Br1215 at a 1:150 dilution, and then stained with secondary and control antibodies (data not shown).

[0099] Incubating a NaV1.5 peptide microarray with plasma from 20 patients with Brugada syndrome (Figure 12) led to the identification of seven autoantibody binding sites on the NaV1.5 channel protein (SEQ ID NO: 1) characterized by the following sequence.

[0100] DI-S5-S6(263-368):SVEADGLVWESLDLYLSDPENYLLKNGTS(Sequence ID 8) DI S5-S6(263-368):CLKAGENPDHGYTSFDSFAWAFLAL(Sequence ID 9) DIII S5-S6(1349-1414):DSDSGLLPRWHMMDFFHAFLII(Sequence ID 10) DIII S5-S6(1349-1414):VNNKSQCESLNLTGEYWTKVK(Sequence ID 11) DIV S3-S4(1598-1634):GSGVILSIVGTVLSDIIQKYFFSPT(Sequence ID 12) DIV S5-S6(1670-1707):MANFAYVKWEAGIDDMFNFQTFANSMLCLF(Sequence ID 13) DIV S5-S6(1711-1754):GWDGLLSPILNTGPPYCDPTLPNSNGSRGD(Sequence ID 14)

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Claims

1. An antibody for use as a biomarker for the diagnosis of Brugada syndrome in humans, wherein the antibody is an antibody against the NaV1.5 channel (SEQ ID NO: 1) and related isoforms.

2. The antibody according to claim 1, for binding sites in the extracellular loop of the NaV1.5 channel (SEQ ID NO: 1) and related isoforms.

3. The antibody according to claim 2, wherein the binding site in the extracellular loop of the NaV1.5 channel and associated isoform is selected from the group comprising the following sequences or fragments thereof: i) VFALIGLQLFMGNLRHKCVRNFTALNGTNGSVEADGLVWESLDLYLS DPENYLLKNGTSDVLLLCGNSSDAGTTCPEGYRCLKAGENPHGGYTSFDSDFAWAFLALFRL (Sequence No. 2) ii) FGKNYSELRDDSDSGLLPRWHMMDFFHAFLIIFRILCG (Sequence ID 3) iii) SIMGVNLFAGKFGRCINQTEGDLPLNYTIVNNKSQCESLNLTGELYW TKVKVNFDNVGAGYLALLQ-1414 (Sequence ID 4) iv)VILSIVGTVLSDIIQKYFFSPTLFRVIRLARIGRI (Sequence ID 5) v) IYSIFGMANFAYVKWEAGIDDMFNFQTFANSMLCLFQI (Sequence ID 6) vi) AGWDGLLSPILNTGPPPYCDPTLPNSNGSRGDCCGSPAVGILFFTT (Sequence No. 7) vii) SVEADGLVWESLDLYLSDPENYLLKNGTS (Sequence No. 8) viiii)CLKAGENPDHGYTSFDSFAWAFLAFLAL (Sequence No. 9) ix) DSDSGLLPRWHMMDFFHAFLII (Sequence No. 10) x) VNNKSQCESLNLTGEYWTKVK (Sequence No. 11) xi)GSGVILSIVGTVLSDIIIQKYFFSPT (Sequence No. 12) xii)MANFAYVKWEAGIDDMFNFQTFANSMLCLF (Sequence ID 13) xiiii)GWDGLLSPILNTGPPYCDPTLPNSNGSRRGD (Sequence No. 14).

4. An in vitro method for detecting the presence or quantity of at least one antibody among antibodies against the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms described in any one of claims 1 to 3 in a biological sample of a person suspected of having Brugada syndrome.

5. a) The step of contacting the biological sample with one or more antigens that specifically bind to the antibody according to any one of claims 1 to 3 for the NaV 1.5 channel (SEQ ID NO: 1) and related isoforms; and b) Step of detecting the binding of the antigen to the antibody in the biological sample. The in vitro method according to claim 4, including the method described in claim 4.

6. The in vitro method according to any one of claims 4 to 5, wherein one or more of the aforementioned antigens are labeled.

7. The in vitro method according to any one of claims 4 to 6, wherein the antigen is a fragment of 10 amino acids or more derived from a binding site in the extracellular loop of a NaV1.5 channel.

8. The in vitro method according to claim 7, wherein the binding site in the extracellular loop of the NaV1.5 channel is selected from SEQ ID NOs: 2 to 14.

9. The in vitro method according to any one of claims 4 to 8, wherein the biological sample is selected from the group consisting of plasma, human peripheral blood mononuclear cells, whole blood, serum and peripheral blood, pericardial fluid, or a combination thereof.

10. The in vitro method according to any one of claims 4 to 9, wherein the detection of the binding of the one or more antigens to the antibody is performed by ELISA, FACS analysis, or Western blotting.

11. An antagonist for use as a therapeutic agent in the treatment of Brugada syndrome, wherein the antibody according to any one of claims 1 to 3 binds to a NaV1.5 channel (SEQ ID NO: 1) and associated isoforms, preferably to one or more antigenic fragments of its extracellular loop.

12. A therapeutic agent for use in the treatment of Brugada syndrome, which specifically targets or interferes with the production of autoantibodies against the NaV1.5 channel (SEQ ID NO: 1) according to any one of claims 1 to 3.

13. A therapeutic agent for use in the treatment of Brugada syndrome according to claim 12, which specifically targets autoantibodies against the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms, characterized by comprising one or more antigenic fragments of the extracellular loop of the NaV1.5 protein.

14. The therapeutic agent according to claim 13, comprising a sequence of five or more amino acids derived from an extracellular loop of the NaV1.5 protein, wherein the extracellular loop is selected from the group consisting of SEQ ID NOs: 2 to 14.

15. An antagonist or therapeutic agent according to any one of claims 11 to 14, for use in combination with another treatment for Brugada syndrome.

16. A kit for detecting antibodies against a NaV1.5 channel in a biological sample, comprising one or more antigens that specifically bind to antibodies against the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms described in any one of claims 1 to 3.

17. The kit according to claim 16, wherein the antigen comprises one or more antigenic fragments of the extracellular loop of the NaV1.5 protein, and the antigenic fragment of the extracellular loop is selected from the group of sequences SEQ ID NOs: 2 to 14.

18. The kit according to any one of claims 16 to 17, wherein the antigen is labeled or bound to a solid support.

19. An antigenic fragment or epitope belonging to a binding site in the extracellular loop of a NaV1.5 channel and associated isoform, selected from the group containing sequences of SEQ ID NOs: 2 to 14.

20. Use of one or more antibodies against the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms according to any one of claims 1 to 3 for the diagnosis of Brugada syndrome in humans by detecting the presence of the antibodies in a biological sample, wherein the biological sample is selected from the group consisting of plasma, human peripheral blood mononuclear cells, whole blood, serum, peripheral blood, pericardial fluid, or a combination thereof.

21. The use according to claim 20, wherein the detection is carried out by using one or more antigens that specifically bind to autoantibodies against the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms.

22. The use according to claim 21, wherein one or more antigens are selected from antigenic fragments or epitopes belonging to binding sites in the extracellular loop of NaV1.5 channels and associated isoforms selected from the group including sequences of SEQ ID NOs: 2 to 14.

23. The use of one or more antibodies according to any one of claims 1 to 3 as markers for monitoring the effectiveness of therapeutic treatment for Brugada syndrome disease by detecting autoantibody titers in biological samples of treated patients.

24. The use of one or more antibodies according to claim 23, wherein the therapeutic procedure is a pharmacological procedure or an interventional surgical procedure including ablation.

25. An animal model obtained by injecting plasma derived from a Brugada patient containing an autoantibody against the NaV1.5 channel (SEQ ID NO: 1) and / or related isoform described in any one of claims 1 to 3, or by injecting one or more antigenic fragments or fragments thereof selected from the group of sequences SEQ ID NO: 2 to 14.

26. The animal model according to any one of claims 25, wherein the animal is a rodent, preferably a mouse.

27. A method for identifying compounds for the prevention and / or treatment of Brugada syndrome, comprising administering a candidate compound to a test animal model according to any one of claims 25 to 26, and detecting a change in antibodies against the NaV1.5 channel (SEQ ID NO: 1) and associated isoforms according to any one of claims 1 to 3 in the test animal model, relative to a control.

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