Auto-antibody aiming at Nav1.5 channel protein as biomarker for diagnosing Brugada syndrome

Abstract

The present invention relates to autoantibodies directed against Nav1.5 channel proteins and related isomers, their use as biomarkers for the diagnosis of human Brugada Syndrome, and related detection and treatment methods targeting such newly discovered autoimmune forms.

Description

Technical Field

[0001] This invention relates to autoantibodies against NaV1.5 channel protein and related isoforms, their use as biomarkers for the diagnosis of human Brugada Syndrome, and related detection and treatment methods targeting this newly discovered form of autoimmunity. Background Technology

[0002] Brugada syndrome (BrS) is a hereditary arrhythmogenic disorder that increases the risk of sudden cardiac death (SCD), accounting for 5-40% of SCD cases in individuals under 40 years of age[1]. The syndrome typically presents as cardiac arrest or syncope and occurs in individuals aged 30 to 40 years[2][3]. However, most patients are asymptomatic and have normal cardiac structure, and these cases are often diagnosed incidentally.

[0003] The diagnosis of Brugada syndrome is based on the presence of dome-shaped ST-segment elevation in the right precordial leads on an electrocardiogram (ECG), which can occur spontaneously or after a stimulatory drug test with an intravenous sodium channel blocker [4]. Although some patients experience cardiac arrest or syncope in their 30 to 40 years of age, most are asymptomatic [2]. Brugada syndrome is a complex disease influenced by both genetic and non-genetic factors [5, 6] involving mutations in more than 23 genes encoding sodium, potassium, and calcium channels and the proteins responsible for their transport [7].

[0004] Among these genes, the α subunit of the voltage-gated sodium channel protein NaV1.5 is encoded. SCN5A It is the main pathogenic gene for Brugada syndrome[8]. However, the genetic cause remains unclear in about 70-75% of cases, and a single mutation cannot fully explain the Brugada syndrome phenotype[9], which makes genetic testing insufficient as an independent diagnostic tool for the disease.

[0005] Brugada syndrome is not solely driven by genetic factors, as evidenced by histological changes in the right ventricular myocardium in patients with type 1 Brugada [10, 11], and by inflammatory infiltration and fibrosis in the right ventricular outflow tract of Brugada patients [12, 13]. Furthermore, emerging evidence suggests a potential role for autoimmunity in Brugada syndrome, a role long overlooked in cardiac arrhythmias. The discovery of autoantibodies associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) reveals the influence of autoimmunity in Brugada syndrome [14, 15].

[0006] Autoantibodies can interfere with ion channel proteins and receptors involved in cardiac electrophysiology, thereby inducing arrhythmias. For example, autoantibodies targeting β1-adrenergic receptors have been reported in a variety of cardiac diseases, including ischemic cardiomyopathy and Chagas disease [16, 17]. In addition, IgG antibodies against voltage-gated KCNQ1 K+ channel proteins (Kv7.1 or KvLQT1) have been detected in patients with dilated cardiomyopathy, resulting in a shortened QTc interval

[18] . Autoantibodies targeting cardiac voltage-gated NA+ channel proteins have also been found in patients with idiopathic high-grade AV block, resulting in a decrease in sodium current (INa) density in rat cardiomyocytes

[19] .

[0007] In summary, further research is needed beyond genetic factors to investigate the role of the immune system in Brugada syndrome in order to fully understand the disease.

[0008] Previous studies have shown abnormal protein distribution in the myocardium of Brugada syndrome, such as α-actin, keratin-24, and connexin-43, which can affect the transport of sodium channel complexes

[12] . These proteins have also been identified as potential targets for autoantibodies, representing abnormal immune responses. Therefore, the presence of NaV1.5 channel autoantibodies in plasma of Brugada syndrome has not yet been definitively proven. Summary of the Invention

[0009] By studying the presence and effects of autoantibodies against the NaV1.5 channel protein, the inventors have now explored the pathophysiology of Brugada syndrome beyond genetic factors.

[0010] In particular, the inventors developed an in vitro model of NaV1.5 overexpressing channel proteins that mimics the physiological environment by improving purification capabilities and the ability to test autoantibody binding under denaturing and native conditions. Previous studies have shown that autoantibodies against NaV1.5 can affect sodium ion currents in rats

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

[0011] By targeting the autoimmune system, these groundbreaking findings offer hope for the development of treatment strategies and improvements in patient care for Brugada syndrome. Mitigating the detrimental effects of autoantibodies on the NaV1.5 channel may contribute to better management of Brugada syndrome and its associated arrhythmias.

[0012] Therefore, one object of the present invention is an antibody for use as a biomarker for the diagnosis of human Brugada syndrome, said antibody targeting the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms.

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

[0014] Therefore, according to the present invention, the term "NaV1.5 channel protein and related isoforms" covers any and all variants of the NaV1.5 channel protein, including but not limited to embryonic isoforms that share significant sequence homology with the classic NaV1.5 channel protein (SEQ ID NO:1). "Significant sequence homology" refers to sequence homology greater than 85%, preferably greater than 90%, and even more preferably greater than 95%, 96%, 97%, 98%, or 99%.

[0015] In a preferred embodiment, the antibody of the present invention used as a biomarker for the diagnosis of human Brugada syndrome targets the extracellular loop binding site of the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms.

[0016] However, it cannot be ruled out that autoantibodies may also target binding sites exposed in the inner channel protein when misfolded protein channels are present in Brugada patients.

[0017] Preferably, the antibody used as a biomarker for the diagnosis of Brugada syndrome in this invention targets an extracellular loop binding site of the NaV1.5 channel protein and related isoforms, wherein the binding site may be selected from the group consisting of the following sequences:

[0018]

[0019] Or a fragment thereof.

[0020] The antibody targets the NaV1.5 channel protein (SEQ ID NO:1), preferably the extracellular loop binding site of the NaV1.5 channel protein, and more preferably the binding site having a sequence selected from SEQ ID NO:2 to SEQ ID NO:14. The antibody is an autoantibody shown in a biological sample of a human affected by Brugada syndrome.

[0021] This invention further relates to an in vitro method for detecting the presence or amount of at least one antibody of the present invention against the NaV1.5 channel protein in a biological sample of a human suspected of having Brugada syndrome. In a preferred embodiment of the invention, the antibody targets the extracellular loop of the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms.

[0022] In a preferred embodiment of the invention, the human may be asymptomatic or at high risk of Brugada syndrome due to family history, previous atrial and / or ventricular fibrillation events, diabetes, or obesity.

[0023] According to a preferred embodiment, the in vitro method includes the following steps:

[0024] a) Contacting a biological sample with one or more antigens, said antigens specifically binding to antibodies against the NaV1.5 channel protein (SEQ ID NO: 1) and related isoforms; and

[0025] b) Detect the binding of the antigen to antibodies in the biological sample.

[0026] Preferably, the one or more antigens in step a) bind to an antibody against the extracellular loop of the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms.

[0027] Preferably, the antigen is a fragment of at least 5 amino acids, more preferably at least 7 amino acids, from the extracellular loop binding site of the NaV1.5 channel protein. In a preferred embodiment, the extracellular loop binding site of the NaV1.5 channel protein is selected from sequences SEQ ID NO:2 to SEQ ID NO:14.

[0028] In a preferred embodiment of the in vitro method for detecting antibodies against NaV1.5 channel protein and related isoforms according to the present invention, the antigen is labeled.

[0029] In a further preferred embodiment of the in vitro method of the present invention, the biological sample is selected from the group consisting of plasma, PBMCs, whole blood, serum, peripheral blood, pericardial fluid, or combinations thereof. Preferably, the biological sample is plasma and / or PBMCs.

[0030] In a preferred embodiment of the in vitro method of the present invention for detecting antibodies against NaV1.5 channel protein and related isoforms, the detection of antigen-antibody binding is performed by ELISA, FACS analysis or immunoblotting.

[0031] A further object of the present invention is to provide an antagonist for antagonizing the binding of antibodies against NaV1.5 channel protein (SEQ ID NO:1) and related isoforms, which can be used as a therapeutic agent for treating Brugada syndrome. Preferably, the antibody targets the extracellular loop of NaV1.5 channel protein (SEQ ID NO:1).

[0032] For example, steroids (such as prednisone, methylprednisolone, and dexamethasone), as well as drugs that can reduce the production of autoantibodies (such as colchicine and hydroxychloroquine), can be advantageously used to treat Brugada syndrome.

[0033] The present invention further contemplates a therapeutic agent specifically targeting an antibody against the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms for the treatment of Brugada syndrome. Preferably, the antibody targets the extracellular loop of the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms.

[0034] According to a preferred embodiment of the invention, the therapeutic agent is characterized in that it comprises one or more antigen fragments of the extracellular loop of the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms.

[0035] Preferably, the autoimmune therapeutic agent comprises a sequence of at least about 10 amino acids from the extracellular loop of the NaV1.5 channel protein, said extracellular loop being selected from the group consisting of sequences SEQ ID NO:2 to SEQ ID NO:14.

[0036] Another possible treatment approach considers targeting B cells to disrupt the production of autoantibodies that can trigger inflammation mediated by immune complexes. Drugs that target B cells and disrupt the production of pathogenic autoantibodies (such as rituximab, ozoglucillin, ofatumumab, and inebilizumab) can be used for therapeutic treatment; these drugs have shown efficacy in treating a variety of B-cell-mediated immune diseases and autoimmune disorders.

[0037] Another treatment for Brugada syndrome involves the use of drugs that inhibit neonatal Fc receptors (FcRn), thereby affecting the recycling and half-life of pathogenic autoantibodies. Nicalimumab, along with other FcRn inhibitors such as efgartigimod, rozanolixizumab, and batoclimab, reduces the level of circulating autoantibodies by preventing their recycling.

[0038] This approach can reduce autoantibody load and mitigate its pathological impact on the NaV1.5 channel. By targeting the FcRn-mediated recirculation pathway, these drugs reduce autoantibody titers without broadly suppressing the immune system, thus providing a safer alternative to traditional immunosuppressive therapy.

[0039] Alternatively, a multivalent or multispecific nanobody-based antibody is considered to target Ab-NaV1.5 to restore normal activity of the NaV1.5 channel. Furthermore, these nanobodies would serve as a valuable tool for in vivo imaging of the cardiac substrate affected by Brugada syndrome, thereby enabling efficient monitoring.

[0040] The aforementioned treatment strategy is based on the significant reduction in sodium current observed in the presence of autoantibodies in mouse models, and the subsequent normalization once the autoantibodies are removed (see [link to treatment strategy]). Figure 9-10 (The data shown).

[0041] The present invention further considers combining antagonists or autoimmune therapies with another therapy for Brugada syndrome to achieve a more comprehensive and effective treatment.

[0042] The present invention further relates to a kit for detecting antibodies against NaV1.5 channel protein (SEQ ID NO:1) and related isoforms in biological samples, the kit comprising one or more antigens that specifically bind to autoantibodies against NaV1.5 channel protein. Preferably, the one or more antigens specifically bind to antibodies against the extracellular loop of NaV1.5 channel protein (SEQ ID NO:1). Even more preferably, the antigen comprises one or more antigen fragments or epitopes belonging to extracellular loop binding sites of NaV1.5 protein, said extracellular loop binding sites being selected from the group consisting of sequences SEQ ID NO:2 to SEQ ID NO:14.

[0043] In a preferred embodiment of the kit of the present invention, the antigen is labeled or attached to a solid support. Preferably, the antibody is fluorescently labeled.

[0044] The solid support is preferably a multi-well plate. Preferably, the kit is an ELISA kit.

[0045] The present invention further relates to antigen fragments or epitopes belonging to the extracellular loop binding site of NaV1.5 channel protein, wherein the extracellular loop binding site is selected from the group consisting of sequences SEQ ID NO:2 to SEQ ID NO:14.

[0046] Furthermore, this invention relates to the use of one or more autoantibodies against the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms for the diagnosis of human Brugada syndrome, the diagnosis being performed by (qualitative or quantitative) detection of the presence of the autoantibody in a biological sample selected from the group consisting of plasma, PBMCs, whole blood, serum, peripheral blood, pericardial fluid, or combinations thereof. According to a preferred embodiment of the invention, the detection step is performed using one or more antigens that specifically bind to the autoantibody against the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms. In a further preferred embodiment of the invention, the one or more antigens are selected from antigen fragments or epitopes belonging to extracellular loop binding sites of the NaV1.5 channel protein, the binding sites being composed of sequences SEQ ID NO:2 to SEQ ID NO:14.

[0047] Furthermore, this invention considers using one or more of the autoantibodies of this invention as biomarkers to monitor the efficacy of treatments for Brugada syndrome by detecting autoantibody titers in biological samples of treated patients. The biological samples may be selected from the group consisting of plasma, PBMCs, whole blood, serum, peripheral blood, peripheral fluid, or combinations thereof. According to a preferred embodiment, the therapeutic treatment may be pharmacological or interventional surgical treatment, including ablation.

[0048] The present invention further relates to an animal model obtained by injecting plasma from a patient affected by Brugada syndrome, the plasma containing autoantibodies against the NaV1.5 channel protein (SEQ ID NO:1) and / or related isoforms disclosed above.

[0049] Alternatively, the present invention relates to an animal model obtained by injection of one or more antigens, said antigens specifically binding to antibodies disclosed above against the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms, said antigen being an antigen fragment selected from the group consisting of sequences SEQ ID NO:2 to SEQ ID NO:14 or fragments thereof.

[0050] In a preferred embodiment of the invention, the animal model is a rodent, preferably a mouse.

[0051] In a preferred embodiment, the animal model is a humanized animal model.

[0052] The "humanized animal model" described in this invention is an animal, typically a rodent (such as a mouse), 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 an antigen that targets a specific antibody, thereby facilitating research on the NaV1.5 channel protein and its associated isoforms in an environment that better represents human biology.

[0053] The present invention further contemplates a method for identifying compounds for the prevention and / or treatment of Brugada syndrome, comprising administering a candidate compound to a test animal model disclosed above, and detecting changes in antibodies against the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms in the test animal model relative to a control. For purposes of non-limiting illustration, the invention will now be described in accordance with preferred embodiments thereof, and particularly with reference to the accompanying drawings. Attached Figure Description

[0054] In the attached diagram:

[0055] Figure 1 The image shows the detection of autoantibodies against the NaV1.5 channel protein in the plasma of patients with Brugada syndrome. Figure A shows the immunoblotting: proteins from lysed cells were separated in one-dimensional SDS-PAGE; a 250-kd NaV1.5 protein band was visible in the lysate of transfected HEK cells (HEK-NaV1.5), but not in the lysate of untransfected (HEK-WT) cells. Anti-rabbit anti-NaV1.5 and anti-human IgG were used to detect the presence and absence of these proteins. SCN5A IgG colocalized in BrS patients but was not detected in healthy controls. Figure B shows immunoprecipitation (IP) of NaV1.5 protein using IgG purified from the plasma of BrS patients and healthy controls (n=3).

[0056] Figure 2 Immunoblot analysis showed that IgG from Brugada syndrome plasma could specifically bind to NaV1.5 in protein lysate from mouse heart tissue.

[0057] Figure 3Representative fluorescence images of NaV1.5 autoantibodies in the plasma of patients with Brugada syndrome are shown. Figure A) shows representative fluorescence images of BrS-positive and negative samples. Red fluorescence highlights NaV1.5 expression, while green fluorescence indicates the presence of bound human antibodies. The same plasma was tested on both transfected and untransfected cells (HEK-WT). Figure B) shows representative fluorescence images of BrS-positive and negative plasma. Red fluorescence indicates NaV1.5 expression, while green fluorescence serves as an indicator of human IgG.

[0058] Figure 4 The immunoblotting analysis of cell lysates of NaV1.5-transfected HEK293A cells prestained with anti-NaV1.5 antibody and subsequently treated with BrS patient plasma and anti-human IgG antibody is shown in Figure A, as well as the immunoblotting analysis of lysates incubated with BrS patient plasma boiled before exposure (Figure B).

[0059] Figure 5 The image shows the detection of autoantibodies against NaV1.5 protein in the plasma of patients with Brugada syndrome at diagnosis (left panel) and after catheter ablation (right panel). Immunoblot analysis: Proteins from lysed cells were separated using one-dimensional SDS-PAGE. A 250-kd NaV1.5 protein band was observed in lysates of transfected HEK cells (HEK-NaV1.5), but not in lysates of untransfected cells (HEK-WT). Autoantibodies against NaV1.5 from cells with and without the protein were investigated using anti-rabbit anti-NaV1.5 antibody and anti-human IgG antibody. SCN5A IgG colocalization in patients with mutated Brugada syndrome but not after ablation (PA).

[0060] Figure 6 This study demonstrates the detection of autoantibodies against NaV1.5 protein in the plasma of patients with Brugada syndrome. Immunoblot analysis: Proteins from lysed cells were separated by one-dimensional SDS-PAGE. A 250-kd NaV1.5 protein band was observed in lysates of transfected HEK cells (HEK-NaV1.5), but not in lysates of untransfected cells (HEK-WT). The presence and absence of autoantibodies were investigated using anti-rabbit anti-NaV1.5 antibody and anti-human IgG antibody. SCN5A IgG colocalization in BrS patients with mutations (rather than healthy controls).

[0061] Figure 7 A comparison of representative fluorescence images of Brugada syndrome-positive and negative plasma is shown. Figure A: Representative fluorescence images showing BrS-positive and negative plasma. Red fluorescence indicates NaV1.5 expression, while green fluorescence serves as an indicator of human IgG. Figure B: Mendes coefficient.

[0062] Figure 8 The effect of autoantibodies from patients with Brugada syndrome on sodium currents is shown. Figure A) Analysis of sodium currents in HEK293A cells overexpressing NaV1.5 incubated with control (CTR, left panel) or Brugada syndrome serum (BrS, right panel, solid circles). Figures BC) Mean current-voltage relationship (IV) (Control: N=6, n=70, hollow circles; BrS serum: N=8, n=91, solid circles).

[0063] Figure 9 The effects of BrS patient plasma on inward currents in hiPSC-derived cardiomyocytes (hiPSC-CM) are shown. Figure A) shows TTX-sensitive sodium current clusters induced by a voltage step protocol after 1 hour of incubation with BrS patient plasma (left) or right, showing a reduction in current density of approximately 50% compared to untreated cardiomyocytes. Figures B and C show the mean current-voltage relationship and voltage dependence of channel activation of TTX-sensitive sodium currents obtained from untreated cells (black triangles, n=26, N=3 different differentiations) or cells incubated with BrS patient plasma (hollow circles, n=33, N=3 plasma samples). (p<0.5). Figure D shows nifedipine-sensitive calcium current clusters in untreated (left) or BrS patient plasma-incubated (right) hiPSC-CM, indicating that the current density is not affected by plasma incubation. Figures E and F show the IV curves and voltage dependence of activation of nifedipine-sensitive sodium-calcium currents in untreated cells (black triangles, n=22, N=3 different differentiations) and cells incubated with BrS patient plasma (n=30, N=3 BrS plasma samples), respectively.

[0064] Figure 10 The electrocardiographic response to plasma in vivo is shown. Figures A and B show ECG recordings of mice before (Figure A) and after (Figure B) administration of BrS patient plasma. Following administration, the ECGs reflected a Brugada-like ECG pattern common in human bipolar leads, characterized by ST-segment elevation in lead III and mirror ST-segment depression in leads I and II. Figures C and D show ECG results in mice treated with plasma from non-BrS control subjects. Notably, no significant ECG changes were observed after infusion (Figure D), indicating the absence of arrhythmogenic activity. Figures E and F show ECG trajectories of mice receiving antibody-depleted BrS plasma. This demonstrates the absence of ECG abnormalities after infusion. Three bipolar lead configurations (leads I, II, and III, respectively) for ECG recording are shown.

[0065] Figure 11The identification of the putative autoantibody binding sites on the NaV1.5 protein is shown. Figure A) Amino acid sequence of the NaV1.5 channel protein (SEQ ID NO:1); Figure B) Six potential regions as autoantibody binding sites were identified. Ten amino acids crucial for binding on a solid support are highlighted in yellow; Figure C) PyMol structure showing the core and extracellular loop of the NaV1.5 channel protein.

[0066] Figure 12 The peptide microarray scans 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) are shown. Detailed Implementation

[0067] Example

[0068] The following examples are merely illustrative and should not be considered as limiting the scope of the invention.

[0069] Example 1 Detection of NaV1.5 channel autoantibodies in plasma of patients with Brugada disease

[0070] method

[0071] Human samples

[0072] This study included Brugada syndrome (BrS) patients diagnosed in the Department of Arrhythmias at the Italian Research Hospital for San Donato (IRCCS Policlinico San Donato)

[24] . Participants were divided into three main subgroups: 1) patients with SCN5A 1) Patients with BrS mutations; 2) Patients without BrS mutations SCN5A Patients with variant BrS; and 3) healthy controls. In addition, five patients underwent catheter ablation. The study protocol 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 a spontaneous or drug-induced type 1 Brugada ECG pattern. Clinical data, medical history, 12-lead ECG recordings, and implantable cardioverter defibrillator (ICD) results were collected from medical records.

[0073] Cell culture

[0074] HEK293 cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum, 1% penicillin-streptomycin and 1% glutamine at 37°C and 5% CO2.

[0075] mouse model

[0076] Adult C57BL-6 mice were housed under standard conditions with a 12-hour light / dark cycle and free access to food and water. All mice were housed in the same controlled environment.

[0077] Whole exome sequencing

[0078] Genes using DNA extracted from peripheral blood (including) SCN5A Next-generation sequencing was performed on all patients. DNA was extracted from peripheral blood and processed to obtain a library containing approximately 575 kb of genomic DNA using a DNA input of 50 nanograms. The library was deep sequenced and, after removing duplicates and filtering low-quality reads, achieved an average target coverage of 100X. Next-generation sequencing data were validated using Sanger sequencing following 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.

[0079] Plasma Collection and Processing

[0080] The blood (25 ml) was centrifuged at 1000g for 15 minutes to separate the plasma, and the supernatant was then centrifuged at 2000g for 15 minutes. The supernatant was collected, aliquoted, and stored at -20°C.

[0081] IgG isolation

[0082] Using PureProteome TM IgG antibodies were separated using a protein G magnetic bead system. Separation was performed under non-denaturing conditions with high salt concentration and near-neutral pH.

[0083] Stable cell line generation and transient transfection

[0084] To express the NaV1.5 channel protein, a protein encoding human... SCN5A The full-length cDNA was obtained and cloned into pcDNA3.1(+). Using ViaFect transfection reagent (Promega), pcDNA3.1(+) / was cloned according to the manufacturer's instructions. SCN5AThe construct was transfected into HEK293 cells. Cells were grown in a medium consisting of Dulbecco modified Eagle medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM glutamine (Merck), and 1X penicillin / streptomycin (Euroclone) and selected using a specific concentration of G418. Cells were maintained at 37°C, 5% CO2, and 95% humidity. Collected cells were lysed, and the clear lysate was collected after centrifugation. Successful transfection and expression of the NaV1.5 channel protein were confirmed by Western blotting analysis.

[0085] Immunoblotting

[0086] Cells were lysed in lysis buffer 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 min. Subsequently, protein (30 µg) 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 rabbit monoclonal anti-NaV1.5 primary antibody (1:2000 dilution, clone D9J7S, Cell Signalling). After washing, the membrane was incubated at room temperature for 1 h with an appropriate anti-rabbit Alexafluor 546 conjugated secondary antibody (1:2000 dilution). After further washing, the membrane was incubated with patient plasma or control plasma diluted 1:3 in PBS at room temperature for 2 hours. After another wash, secondary antibody against human IgG-FITC was added and incubated at room temperature for 1 hour. Immunoreactive bands were visualized using an enhanced chemiluminescence detection kit (ECL Advance, GE Healthcare).

[0087] NaV1.5 immunoblotting

[0088] HEK293A cells overexpressing NaV1.5 were lysed using RIPA buffer containing a mixture of protease and phosphatase inhibitors, followed by centrifugation at 15,000 rpm for 10 min at 4 °C to collect the supernatant. Protein concentration was determined by BCA assay (Pierce). The protein was denatured and reduced in Laemmli buffer containing β-mercaptoethanol (Bio-Rad), and loaded onto a 10% SDS-PAGE gel (Protean Tgx stain-free, Bio-Rad) for electrophoresis, then transferred to a nitrocellulose membrane. After blocking, the membrane was probed overnight at 4 °C with anti-NaV1.5 primary antibody (1:2000 dilution, Cell Signaling, clone D9J7S), followed by washing and incubation with anti-rabbit IRDye 800 CW secondary antibody (1:2000 dilution, LI-COR Biosciences). The membrane was then incubated with plasma from BrS patients that had been boiled at 100°C for 10 minutes or untreated, followed by staining with anti-human IgG-HRP secondary antibody (1:2000 dilution, Bio-Rad). Bands were visualized using an ECL Advance kit (GE Healthcare) and imaged using a ChemiDoc MP system (Bio-Rad).

[0089] Immunoprecipitation of NaV1.5 channel protein from protein lysate of mouse heart tissue

[0090] To collect cardiac samples, animals were anesthetized by intraperitoneal injection of 0.5 mg / kg medetomidine (OrionPharma Srl) and 100 mg / kg ketamine (Merial) (both diluted in physiological saline). After the animals were fully unconscious, their chests were opened, and the heart was perfused with 1 ml of 1M KCl to induce diastolic arrest. The heart was then flushed with 0.9% physiological saline 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 sectioned into 3 mm thick slices using a specific stainless steel heart matrix (Roboz Surgical Instruments) to produce samples from the apex, middle, and base of the heart. The apex was incubated with 500 ml of RIPA lysis buffer and homogenized using a Lyser® tissue homogenizer (Qiagen). Each sample underwent three homogenization cycles, each lasting 5 minutes, at a frequency of 25 oscillations per second. The homogenate was placed 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.

[0091] 300 μg of total protein was mixed with Dynabeads containing IgG bound to BrS plasma or a control. TM Protein G was incubated, and then, as described above, the eluted fraction was subjected to immunoblotting analysis for NaV1.5 staining.

[0092] Immunoprecipitation of NaV1.5 from transfected HEK-293 cells.

[0093] Immunoprecipitation of NaV1.5 channel protein in transfected HEK-293 cells was performed using IgG specific to NaV1.5 channel protein from patient serum and healthy control serum. Immunoprecipitation was performed by incubating the antibody with Dynabeads protein G at room temperature for 30 minutes. After washing, the beads were incubated with total cell lysate at room temperature with agitation for 2 hours. After further washing, Laemmli-β-mercaptoethanol sample buffer was added to the beads, and the elution fraction was subjected to SDS-PAGE.

[0094] Mouse tissue immunofluorescence

[0095] Immunofluorescence assays were performed on cardiac tissue from adult C57BL-6 mice. 12 µm thick left ventricular sections were prepared using a cryostat and mounted on gelatin-coated histological slides. Sections were thawed, rehydrated, and then exposed to antigens. Nonspecific binding was blocked using a solution containing normal donkey serum and bovine serum albumin. Sections were incubated with patient plasma diluted 1:50 in PBS (containing 2% NDS and 2% BSA), washed, and then incubated with anti-human IgG-FITC secondary antibody. Subsequently, sections were incubated with rabbit monoclonal anti-NaV1.5 primary antibody (1:200 dilution; clone D9J7S, Cell Signaling), washed, and then incubated with an appropriate Cy3-conjugated anti-rabbit secondary antibody. After further washing, sections were mounted with Vectashield mounting medium containing DAPI. Images were captured using a Leica Thunder microscope (×40 objectives).

[0096] Cell-based immunofluorescence assay (CBA-IF)

[0097] To assess the binding of IgG-NaV1.5 autoantibody, live HEK293 WT cells and HEK293 cells expressing NaV1.5 protein were used. Cells were seeded on coverslips and incubated with patient plasma diluted 1:3 in DMEM HG (supplemented with 1% L-glutamine, 1% penicillin-streptomycin, and 5% heat-inactivated serum). After washing, cells were fixed with cold methanol and blocked. Cells were incubated with NaV1.5 antibody, washed, and stained with rabbit secondary antibody and FITC anti-human IgG. Nuclear staining was performed using DAPI. Images were captured using a Leica Thunder microscope (×40x objective).

[0098] Co-location Quantitative

[0099] Confocal microscopy images were randomly captured for all groups, and colocalization analysis was performed using the JACoP plugin in FIJI software. The Mendes coefficient was calculated to quantify colocalization.

[0100] Cellular electrophysiology

[0101] Sodium currents in HEK293A cells transiently transfected with NaV1.5 channels were recorded using a chip-based automated planar patch-clamp system, Patchliner (Nanion Technologies GmbH, Munich, Germany). Cells were incubated for 1 hour at 37°C and 5% CO2 with 5% BrS or control-derived serum, then gently digested with trypsin and resuspended in Nanion's extracellular low-sodium recording solution (catalog number 08-3004, ionic composition: 80 mM NaCl, 60 mM NMDG, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM D-glucose monohydrate, 10 mM Hepes; pH adjusted to 7.4 with HCl, 289 mOsm). Untreated cells were used as internal controls. To improve membrane stability, cells were then incubated at 4°C for 20 minutes. All recordings were performed at room temperature in a whole-cell configuration using a medium-resistance NPC-16 chip. At least two chips were used per experimental day for each condition. CsF-based intracellular solutions (catalog number 08-3008, ionic composition: 10 mM EGTA, 10 mM Hepes, 10 mM CsCl, 10 mM NaCl, 110 mM CsF; pH adjusted to 7.2 with CsOH, 280 mOsm) and extracellular sealing enhancement solutions (catalog number 08-3011, ionic composition: 80 mM NaCl, 60 mM NMDG, 4 mM KCl, 10 mM CaCl2, 1 mM MgCl2, 5 mM D-glucose monohydrate, 10 mM Hepes; pH adjusted to 7.4 with HCl, 313 mOsm) were also provided by Nanion. To minimize arbitrary bias due to transfection variability, at least one BrS serum sample and one control serum sample from a healthy donor were tested each experimental day. Cell capacitance and series resistance were automatically compensated by Patchliner. All currents were sampled at 50 kHz. The current-voltage (IV) relationship and activation voltage dependence of sodium current were obtained using a scheme with 50 ms increments ranging from -80 mV to +60 mV (holding potential -120 mV). Raw trajectories recorded by the HEK293A amplifier were exported using a self-built Python tool, and individual trajectories were analyzed using Clampfit 10.7 (Molecular Devices, San Jose, CA, USA), Origin Pro (OriginLab, Northampton, MA, USA), and GraphPad Prism (GraphPad Software, Boston, MA, USA). Current density was calculated by dividing the current amplitude (pA) per cell by the cell capacitance (pF).

[0102] Current measured in hiPSC-CM

[0103] At 37°C, in a whole-cell configuration, an inward current was induced manually via patch-clamp, using a stepwise approach from -80 mV to 60 mV for 150 ms (holding potential -80 mV). This approach 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 by subsequent subtraction during analysis. Intracellular solution: 135 mM CsCl, 10 mM NaCl, 5 mM EGTA, 2 mM CaCl2, 2 mM TEA-Cl, 10 mM HEPES, 2 mM MgATP; pH adjusted to 7.2 with CsOH. Extracellular solution: 80 mM NaCl.

[0104] Animals and Electrocardiogram

[0105] Procedures involving mice were performed according to the Animal Experimentation Protocol Guidelines of the Institutional Animal Care and Use Committee (IACUC) of the San Raffaele Scientific Institute (Milan, Italy), authorization number 425 / 2022 / PR. 50-week-old C57BL-6 mice were maintained at room temperature with free access to water and a standard diet, using a 12-hour light / dark schedule. They were anesthetized by intraperitoneal injection of 0.5 mg / kg metopril (Orion Pharma Srl) and 100 mg / kg ketamine (Merial) (both diluted in physiological saline). Body weight was measured prior to each study, and body temperature was continuously monitored using a thermostat system with a rectal thermometer probe (Harvard Apparatus, Holliston, MA, USA) and maintained at 37 ± 0.5 °C. 200 µl plasma of BrS (n=4) and CTR (n=3) was preheated at 56 °C for 20 minutes and then intravenously injected into the anesthetized mice.

[0106] In summary, ECGs were performed continuously 10 minutes after anesthesia induction and 30 minutes after plasma infusion, using four subcutaneous needle electrodes (stainless steel, 27 gauge, 12 mm long; SEI EMG srl, Cittadella, Italy): two needles were inserted into the forelimb, one into the left hindlimb, and the third needle electrode was placed in the right hindlimb as ground. The lead configuration was kept consistent before, during, and after plasma administration, without altering the polarity or placement of the subcutaneous needle electrodes. Specifically, leads were placed in mice using the standard Einthoven configuration, ensuring the same procedure was performed for each mouse to obtain accurate results. The lead configuration remained unchanged throughout the plasma infusion challenge. The following is a general description of ECG lead placement in mice:

[0107] - Lead I: This lead measures the potential difference between the right and left forearms (or right and left forearms). Electrodes are placed on both the right forearm (negative) and left forearm (positive). This is shown in the first line of each ECG test.

[0108] - Lead II: This lead measures the potential difference between the right forelimb (negative electrode) and the left hindlimb (positive electrode). Electrodes are placed on the right forelimb and left hindlimb. This is shown in the second line of each ECG test.

[0109] - Lead III: This lead measures the potential difference between the left forelimb (negative electrode) and the left hindlimb (positive electrode). Electrodes are placed on the left forelimb and left hindlimb. This is shown in the third line of each ECG test.

[0110] - Grounding electrode: The grounding electrode is placed in the neutral area (i.e., the right hind leg) to stabilize the signal and reduce noise.

[0111] The needle electrode was connected to an amplifier (Micromed, Moriano Veneto, Italy) via a flexible cable, and the ECG signal was recorded using System-Plus software (Micromed, Moriano Veneto, Italy) at 256 Hz (16-bit), with bandpass filters from 1 Hz to 70 Hz. All animal experiments were performed without prior knowledge of the plasma sample source to ensure the integrity of the results.

[0112] result

[0113] Detection of autoantibodies against NaV1.5 protein in plasma of patients with Brugada syndrome

[0114] This study included a cohort of 100 participants. Initially, immunoblotting analysis was performed to detect the presence of IgG autoantibodies against the NaV1.5 channel protein in patient plasma samples. Of the BrS patients (n=53) who tested positive in the ajmaline test, 48 did not have... SCN5A Patients with mutations and 5 carriers SCN5A Patients with the variant exhibited this autoantibody. Conversely, plasma samples from 47 subjects who tested negative in the amalin trial did not show the presence of this autoantibody. Figure 1 Figure A in the middle, and Figure 6 To further confirm the presence of IgG targeting the NaV1.5 channel protein, an immunoprecipitation assay was performed. IgG isolated from the plasma of patients with positive autoantibodies showed binding to NaV1.5 protein extracted from HEK cell lysates. Figure 1 (Figure B in the text).

[0115] Mouse NaV1.5 was immunoprecipitated with BrS anti-NaV1.5 autoantibody.

[0116] Immunoblotting analysis showed that IgG from BrS plasma could specifically bind to NaV1.5 in protein lysate from mouse heart tissue. Figure 2 ).

[0117] Magnetic beads coated with IgG from BrS patients were incubated with mouse ventricular protein extracts. Subsequently, immunoblotting analysis was used to resolve the immunoprecipitation (IP) fraction and the immune exhaustion (I-) fraction. Figure 2 (Middle left image).

[0118] The presence of NaV1.5 protein in the IP fraction (discovered by staining with a commercially available anti-NaV1.5 antibody) Figure 2 The right-middle figure confirms the presence of anti-NaV1.5 IgG in BrS plasma.

[0119] Plasma IgG from BrS patients binds to NaV1.5 on cells overexpressing NaV1.5 channel protein.

[0120] To investigate the binding of plasma IgG from BrS patients to NaV1.5, dual immunofluorescence labeling was performed on mouse heart sections using an anti-NaV1.5 monoclonal antibody and plasma from BrS patients and healthy controls. Colocalization of immunofluorescence signals was observed when using the monoclonal anti-NaV1.5 antibody and BrS patient plasma, but no signal was observed using healthy control plasma. Figure 3 (See Figure A in the figure). Similar results were obtained by immunolabeling cells overexpressing NaV1.5 channel protein using plasma from BrS patients. Anti-NaV1.5 antibodies from BrS patients specifically bound to NaV1.5-transfected cells, but not to cells with simulated transfection. Figure 3 Figure B and Figure 7 ).

[0121] Furthermore, immunoblotting analysis of cell lysates from NaV1.5-transfected HEK293A cells pre-stained with anti-NaV1.5 antibody and subsequently treated with BrS patient plasma and anti-human IgG antibody confirmed the specific interaction between BrS patient autoantibodies and NaV1.5. Figure 4 (Figure A).

[0122] The same experiment, performed after incubation of BrS patient plasma boiled before exposure with lysis buffer, showed loss of binding of specific anti-NaV1.5 IgG to NaV1.5 protein, indicating that thermal denaturation of IgG abolishes its binding ability. Figure 4 (Figure B).

[0123] No autoantibodies against NaV1.5 were detected in BrS patients after catheter ablation.

[0124] To assess the impact of catheter ablation on the presence of autoantibodies against NaV1.5, a subgroup of five patients from the initial cohort was analyzed six months post-ablation. Immunoblot analysis showed that these patients lacked IgG autoantibodies against NaV1.5 after epicardial ablation. Figure 5 ).

[0125] The effect of autoantibodies on sodium current in patients with Brugada syndrome

[0126] The inventors conducted in vitro experiments to evaluate the effect of IgG removal on sodium currents in HEK293 cells and hiPSC-derived cardiomyocytes (see below).

[0127] in particular, Figure 8 The study demonstrated that autoantibodies in patients with Brugada syndrome reduced sodium currents. A fully configured automated patch-clamp was used to record sodium currents in HEK293 cells overexpressing the NaV1.5 channel protein, incubated for 1 hour with 5% BrS patient plasma or plasma from a healthy donor.

[0128] The results showed that the current density of the former was significantly reduced by approximately 40% compared to -211.2 ± 21 pA / pF (n = 79) measured under the latter condition, with a mean peak current density of -122.3 ± 12 pA / pF (n = 100) at -20 mV. As an internal control, the current density recorded in untreated HEK293 cells overexpressing NaV1.5 was 169.8 ± 12 (n = 54), which was not different from the current density obtained in cells incubated with healthy donor plasma. The data indicate that exposure to Brugada syndrome plasma resulted in a significant reduction (approximately 40%) in inward sodium current compared to control plasma.

[0129] Effects of plasma from patients with Brugada syndrome on inward currents in hiPSC-derived cardiomyocytes (hiPSC-CM)

[0130] Figure 9 Figures A through F in the figure show the effect of Brugada syndrome plasma on the inward current of hiPSC-CM. These results strongly suggest the specificity of the effect of BrS plasma on sodium current.

[0131] Effects of BrS patient plasma injection on wild-type mice

[0132] To further demonstrate the functional role of autoantibodies against the NaV1.5 channel protein in the pathophysiology of Brugada syndrome, the inventors conducted in vivo experiments on adult C57BL-6 mice to evaluate the effect of IgG on sodium current.

[0133] Electrophysiological characteristics of mice were assessed using ECG. Seven mice expressing wild-type NaV1.5 channel protein isoforms were continuously monitored via ECG after intravenous administration of plasma from four BrS patients and control subjects under general anesthesia. Mice receiving BrS plasma developed Brugada ECG ST-segment abnormalities, followed by complex malignant arrhythmias (ventricular arrhythmias and atrioventricular [AV] block), leading to cardiac arrest and death. Figure 10 (Figure AB in the diagram).

[0134] However, mice receiving control plasma did not show ECG changes, including no ST-segment elevation or conduction disturbances. Figure 10 (See Figures C and D in the original text). Furthermore, removing autoantibodies from the plasma prior to infusion prevented the Brugada-like phenotype in mice. Figure 10 Figure EF in the figure. Previously, administration of the same BrS plasma containing autoantibodies resulted in a fornix-shaped ST-segment elevation pattern and a malignant arrhythmia phenotype (Figure EF in the figure). Figure 10 (See Figures AB in the original text). This evidence supports the claim that the treatment methods and / or drugs described in this invention, which can reduce autoantibody levels, can mitigate the risk of autoantibody-induced arrhythmias.

[0135] Example 2 Identification of the putative NaV1.5 autoantibody binding site on the NaV1.5 channel protein

[0136] The human NaV1.5 channel protein has the following amino acid sequence:

[0137]

[0138]

[0139] Prediction of NaV1.5 autoantibody binding sites on NaV1.5 channel proteins

[0140] To explore the binding site of autoantibodies on the NaV1.5 channel protein, molecular modeling was performed using the crystal structure of the NaV1.5 channel protein downloaded from Uniprot SCN5A. Figure 11 Pymol software was used to predict favorable interactions between autoantibodies and target proteins, thereby promoting the rational design of binding sites.

[0141] Epitope discovery

[0142] The sequences of rings 1 to 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 at the N-terminus and C-terminus using neutral GGSGSGSG linkers (SEQ ID NO:15) to avoid truncating the peptides. The extended sequences were converted into 15-amino acid peptides with peptide-peptide overlap of 14 amino acids. The resulting NaV1.5 peptide microarray contained 327 different peptides, which were printed in a repeating manner (654 peptide dots) and framed with additional HA (YPYDVPDYAG SEQ ID NO:16, 40 dots) and poliomyelitis (KEVPALTAVETGAT SEQ ID NO:17, 38 dots) control peptides.

[0143] The microarray was treated with Rockland blocking buffer MB-070 for 30 minutes, and then pre-stained with secondary antibody goat anti-human IgG (Fc) DyLight680 (0.1 µg / ml) in incubation buffer for 45 minutes at room temperature to investigate background interactions that may interfere with the primary assay.

[0144] Further NaV1.5 peptide microarrays were incubated with 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) at a 1:150 dilution in incubation buffer (PBS, pH 7.4, containing 0.05% Tween 20) at 4°C with a rocking motion at 140 rpm for 16 hours. The microarrays were washed three times with 10% Rockland blocking buffer MB-070 for 10 seconds each time, followed by staining with secondary antibody.

[0145] Readouts were performed using an Innopsys InnoScan 710-IR microarray scanner. Additional HA peptides in the framed peptide microarray were simultaneously stained with mouse monoclonal antibody HA (12CA5) DyLight800 (0.2 µg / ml) in PBS (pH 7.4, containing 0.05% Tween 20) at room temperature for 45 minutes (washed three times for 10 seconds each with 10% Rockland blocking buffer MB-070).

[0146] Quantification of spot intensities and peptide annotation were based on a 16-bit grayscale TIFF file, which exhibits a higher dynamic range than the 24-bit color TIFF file shown in this report. Microarray image analysis was performed using PepSlide® Analyzer, a software algorithm that decomposes the fluorescence intensity of each spot into the original signal, foreground signal, and background signal, and calculates the mean median foreground intensity. Intensity maps were generated based on the mean median foreground intensity, and interactions in the peptide map were highlighted by intensity color coding, where red represents high spot intensities and white represents low spot intensities. An Innopsys InnoScan 710-IR microarray scanner with a resolution of 20 µm was used; the scan gain was 50 at low laser power (680 nm, red) and 10 at high laser power (800 nm, green).

[0147] Using molecular modeling, the inventors identified six (DI-DVI) putative autoantibody binding sites on the extracellular loop of the NaV1.5 channel protein (shown in bold in SEQ ID NO:1), as follows: Figure 11 As shown:

[0148]

[0149] Epitope identification in human plasma of patients with Brugada disease

[0150] The NaV1.5 peptide microarray was incubated with a 1:150 dilution of human plasma Dg2677, Dg2818, Br1200, Dg2675, Dg1936, Br1211, Dg2821, Dg2533, Dg2429, Dg2388, Dg2830, Dg2655, Dg2828, Br1196, Dg2720, Dg2819, Br1145, Br938, Br1204, and Br1215, and then stained with secondary antibody and control antibody (data not shown).

[0151] Incubation of NaV1.5 peptide microarray with plasma from 20 patients with Brugada syndrome ( Figure 12 This led to the identification of seven autoantibody binding sites on the NaV1.5 channel protein (SEQ ID NO:1), characterized by the following sequence:

[0152]

[0153] References

[0154] 1. Antzelevitch, C. and B. Patocskai, Brugada Syndrome: Clinical, Genetic, Molecular, Cellular, and Ionic Aspects.Curr Probl Cardiol, 2016. 41(1): p. 7-57.

[0155] 2. Nademanee, K., et al., Fibrosis, Connexin-43, and Conduction Abnormalities in the Brugada Syndrome. J Am Coll Cardiol, 2015. 66(18): p.1976-1986.

[0156] 3. Priori, SG, et al., 2015 ESC Guidelines for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death: The Task Force for the Management of Patients with Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death of the European Society of Cardiology (ESC). Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC). Eur Heart J, 2015. 36(41): p. 2793-2867.

[0157] 4. Batchvarov, VN, The Brugada Syndrome - Diagnosis, Clinical Implications and Risk Stratification. Eur Cardiol, 2014. 9(2): p. 82-87.

[0158] 5. Ghiroldi, A., et al., Alterations of the Sialylation Machinery in Brugada Syndrome. Int J Mol Sci, 2022. 23(21).

[0159] 6. Keller, DI, et al., Brugada syndrome and fever: genetic and molecular characterization of patients carrying SCN5A mutations. CardiovascRes, 2005. 67(3): p. 510-9.

[0160] 7. Barc, J., et al., Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility. Nat Genet, 2022. 54(3): p. 232-239.

[0161] 8. Sonoda, K., et al., Copy number variations of SCN5A in Brugada syndrome. Heart Rhythm, 2018. 15(8): p. 1179-1188.

[0162] 9. Wijeyeratne, Y.D., et al., SCN5A Mutation Type and a Genetic Risk Score Associate Variably With Brugada Syndrome Phenotype in SCN5A Families. Circ Genome Precis Med, 2020. 13(6): p. e002911.

[0163] 10. Frustaci, A., et al., Cardiac histological substrate in patients with clinical phenotype of Brugada syndrome. Circulation, 2005. 112(24): p.3680-7.

[0164] 11. Corrado, D., et al., Relationship Between Arrhythmogenic Right Ventricular Cardiomyopathy and Brugada Syndrome: New Insights From Molecular Biology and Clinical Implications. Circ Arrhythm Electrophysiol, 2016. 9(4):p. e003631.

[0165] 12. Miles, J., et al., Biventricular Myocardial Fibrosis and Sudden Death in Patients With Brugada Syndrome. J Am Coll Cardiol, 2021. 78(15): p.1511-1521.

[0166] 13. Pieroni, M., et al., Electroanatomic and Pathologic Right Ventricular Outflow Tract Abnormalities in Patients With Brugada Syndrome. JAm Coll Cardiol, 2018. 72(22): p. 2747-2757.

[0167] 14. Chatterjee, D., et al., An autoantibody identifies arrhythmogenic right ventricular cardiomyopathy and participates in its pathogenesis. EurHeart J, 2018. 39(44): p. 3932-3944.

[0168] 15. Meraviglia, V., et al., Inflammation in the Pathogenesis of Arrhythmogenic Cardiomyopathy: Secondary Event or Active Driver? FrontCardiovasc Med, 2021. 8: p. 784715.

[0169] 16. Patel, PA and AF Hernandez, Targeting anti-beta-1-adrenergic receptor antibodies for dilated cardiomyopathy. Eur J Heart Fail, 2013. 15(7): p. 724-9.

[0170] 17. Labovsky, V., et al., Anti-beta1-adrenergic receptor autoantibodies in patients with chronic Chagas heart disease. Clin ExpImmunol, 2007. 148(3): p. 440-9.

[0171] 18. Li, J., et al., Anti-KCNQ1 K(+) channel autoantibodies increase IKs current and are associated with QT interval shortening in dilated cardiomyopathy. Cardiovasc Res, 2013. 98(3): p. 496-503.

[0172] 19. Korkmaz, S., et al., Provocation of an autoimmune response to cardiac voltage-gated sodium channel NaV1.5 induces cardiac conduction defects in rats. J Am Coll Cardiol, 2013. 62(4): p. 340-9.

[0173] 20. Chatterjee, D., et al., An autoantibody profile detects Brugada syndrome and identifies abnormally expressed myocardial proteins. Eur HeartJ, 2020. 41(30): p. 2878-2890.

[0174] 21. Mader, S., et al., Complement activating antibodies to myelin oligodendrocyte glycoprotein in neuromyelitis optica and related disorders. JNeuroinflammation, 2011. 8: p. 184.

[0175] 22. Duong, S.L. and H. Pruss, Molecular disease mechanisms of human antineuronal monoclonal autoantibodies. Trends Mol Med, 2023. 29(1): p. 20-34.

[0176] 23. Kobayashi, S., et al., Autoantibody-induced internalization of nicotinic acetylcholine receptor alpha3 subunit exogenously expressed in human embryonic kidney cells. J Neuroimmunol, 2013. 257(1-2): p. 102-6.

[0177] 24. Pappone, C. and V. Santinelli, Brugada Syndrome: Progress in Diagnosis and Management. Arrhythm Electrophysiol Rev, 2019. 8(1): p. 1-6pm.

[0178] 25. Pappone, C., et al., Assessing the Malignant Ventricular Arrhythmic Substrate in Patients With Brugada Syndrome. J Am Coll Cardiol, 2018. 71(15): p. 1631-1646.

[0179] 26. Pappone, C., et al., Electrical Substrate Elimination in 135 Consecutive Patients With Brugada Syndrome. Circ Arrhythm Electrophysiol, 2017. 10(5): p. e005053.

[0180] 27.Richards, S., et al., Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med, 2015. 17(5): p. 405-24

Claims

1. An antibody used as a biomarker for the diagnosis of human Brugada syndrome, said antibody targeting the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms.

2. The antibody according to claim 1, which targets the extracellular loop binding site of the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms.

3. The antibody according to claim 2, wherein the extracellular loop binding site of the NaV1.5 channel protein and related isoforms is selected from the group consisting of the following sequences: Or a fragment thereof.

4. An in vitro method for detecting the presence or amount of an antibody against the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms in a biological sample of a human suspected of having Brugada syndrome.

5. The in vitro method according to claim 4, comprising the following steps: a) Contacting a biological sample with one or more antigens, said antigens specifically binding to the antibody against the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms as described in any one of claims 1-3; and b) Detect the binding of the antigen to the antibody in the biological sample.

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

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

8. The in vitro method according to claim 7, wherein the extracellular loop binding site of the NaV1.5 channel protein is selected from SEQ ID NO:2 to SEQ ID NO:

14.

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

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

11. An antagonist, which is an antagonist of the antibody of any one of claims 1-3 that binds to the NaV1.5 channel protein (SEQ ID NO: 1) and related isoforms, preferably to one or more antigen fragments of its extracellular loop, said antagonist being used as a therapeutic agent for treating Brugada syndrome.

12. A therapeutic agent that specifically targets or disrupts the production of autoantibodies against the NaV1.5 channel protein (SEQ ID NO: 1) according to any one of claims 1-3, for the treatment of Brugada syndrome.

13. The therapeutic agent for treating Brugada syndrome according to claim 12, wherein the agent specifically targets an autoantibody against the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms, characterized in that... The therapeutic agent contains one or more antigen fragments of the extracellular loop of the NaV1.5 protein.

14. The therapeutic agent according to claim 13, characterized in that... The therapeutic agent comprises a sequence of at least 5 amino acids from the extracellular loop of the NaV1.5 protein, wherein the extracellular loop is selected from the group consisting of SEQ ID NO:2 to SEQ ID NO:

14.

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

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

17. The kit of claim 16, wherein the antigen comprises one or more antigen fragments of the extracellular loop of NaV1.5 protein, the antigen fragments of the extracellular loop being selected from the group consisting of sequences SEQ ID NO:2 to SEQ ID NO:

14.

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

19. An antigen fragment or epitope belonging to the extracellular loop binding site of NaV1.5 channel protein and related isoforms, selected from the group comprising the sequences SEQ ID NO:2 to SEQ ID NO:

14.

20. Use of one or more antibodies against NaV1.5 channel protein (SEQ ID NO:1) and related isoforms according to any one of claims 1-3 for the diagnosis of human Brugada syndrome, said diagnosis being performed by detecting the presence of said antibody in a biological sample, said biological sample being selected from the group consisting of plasma, PBMCs, whole blood, serum, peripheral blood, pericardial fluid, or combinations thereof.

21. The use according to claim 20, wherein the detection is performed using one or more antigens that specifically bind to an autoantibody against the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms.

22. The use according to claim 21, wherein the one or more antigens are selected from antigen fragments or epitopes belonging to extracellular loop binding sites of NaV1.5 channel proteins and related isoforms, and are selected from the group comprising the sequences SEQ ID NO:2 to SEQ ID NO:

14.

23. Use of one or more antibodies according to any one of claims 1-3, as biomarkers for monitoring the efficacy of therapeutic treatment for Brugada syndrome 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 treatment is a pharmaceutical treatment or an interventional surgical treatment, including ablation.

25. An animal model obtained by injection of plasma from a patient with Brugada, the plasma containing an autoantibody against the NaV1.5 channel protein (SEQ ID NO:1) and / or related isoforms according to any one of claims 1-3, or the animal model obtained by injection of one or more antigen fragments selected from the group consisting of sequences SEQ ID NO:2 to SEQ ID NO:14 or fragments thereof.

26. The animal model according to claim 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, the method comprising administering a candidate compound to a test animal model according to any one of claims 25-26, and detecting changes in antibodies against the NaV1.5 channel protein (SEQ ID NO:1) and related isoforms according to any one of claims 1-3 in the test animal model relative to a control.

Images