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WO2025021888A1 - Auto-anticorps dirigés contre le canal nav1.5 en tant que biomarqueurs pour le diagnostic du syndrome de brugada - Google Patents

Auto-anticorps dirigés contre le canal nav1.5 en tant que biomarqueurs pour le diagnostic du syndrome de brugada Download PDF

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WO2025021888A1
WO2025021888A1 PCT/EP2024/071047 EP2024071047W WO2025021888A1 WO 2025021888 A1 WO2025021888 A1 WO 2025021888A1 EP 2024071047 W EP2024071047 W EP 2024071047W WO 2025021888 A1 WO2025021888 A1 WO 2025021888A1
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seq
channel
nav
antibodies
anyone
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Luigi Anastasia
Adriana Tarantino
Andrea Ghiroldi
Giuseppe CICONTE
Carlo Pappone
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Cardiomix SRL
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Cardiomix SRL
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Priority claimed from EP23187367.0A external-priority patent/EP4498088A1/fr
Priority claimed from US18/387,237 external-priority patent/US20250145704A1/en
Priority claimed from EP24161814.9A external-priority patent/EP4614149A1/fr
Application filed by Cardiomix SRL filed Critical Cardiomix SRL
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/326—Arrhythmias, e.g. ventricular fibrillation, tachycardia, atrioventricular block, torsade de pointes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates

Definitions

  • the present invention relates to autoantibodies against NaVl.5 channel and related isoforms, their use as biomarker for the diagnosis of Brugada syndrome in human beings and relative methods of detection and therapeutic approach to target this new discovered form of autoimmunity.
  • the Brugada Syndrome is an inherited arrhythmogenic disease that poses an increased risk of sudden cardiac death (SCD), accounting for 5-40% of SCD cases in individuals under 40 years of age [1], The syndrome typically manifests with cardiac arrest or syncope, occurring in the third and fourth decade of life [2] [3] however, the majority of patients are asymptomatic with structurally normal hearts, and they are usually diagnosed incidentally.
  • SCD sudden cardiac death
  • ECG electrocardiogram
  • SCN5A which encodes the alpha subunit of the voltage-gated sodium channel NaV1.5, is the primary causative gene for Brugada syndrome [8],
  • the genetic etiology remains unclear in approximately 70-75% of cases, and a single mutation does not fully account for the Brugada syndrome phenotype [9], rendering genetic testing inadequate as a standalone diagnostic tool for the disease.
  • Brugada syndrome is not solely driven by genetic factors, as evidenced by histological changes in the right ventricular myocardium of type 1 Brugada patients [10, 11] and the presence of inflammatory infiltrates and fibrosis in the right ventricular outflow tract of Brugada patients has been reported [12, 13], Furthermore, emerging evidence suggests a potential role of autoimmunity in Brugada syndrome, which has long been overlooked in cardiac arrhythmias.
  • the discovery of autoantibodies associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) has shed light on the autoimmune implications in Brugada syndrome [14, 15],
  • Autoantibodies can interfere with ion channels and receptors involved in cardiac electrophysiology, thereby triggering arrhythmias.
  • autoantibodies targeting pi -adrenergic receptors have been reported in various cardiac diseases, including ischemic cardiomyopathy and Chagas disease [16, 17]
  • IgG antibodies against the voltage-gated KCNQ1 K+ channel Kv7.1 or KvLQTl
  • Kv7.1 or KvLQTl voltage-gated KCNQ1 K+ channel
  • dilated cardiomyopathy resulting in a shortened QTc interval
  • Autoantibodies targeting the cardiac voltage-gated Na+ channel have also been found in patients with idiopathic high-degree AV block, leading to reduced sodium current (INa) density in rat cardiomyocytes [19]
  • the inventors have developed an in-vitro model of NaV1.5 overexpressing channels with improved purification capabilities and the ability to test autoantibody binding under denaturing and native conditions, mimicking the physiological environment.
  • Previous studies have shown that autoantibodies against NaVl.5 can affect sodium ion currents in vivo in rats [20], This mechanism aligns with the activity of autoantibodies against channel or receptor proteins found in other diseases, such as NMDAR encephalitis, where autoantibodies promote receptor internalization and induce electrophysiological changes in neurons [21- 23],
  • NaV1.5 channel and related isoforms encompasses any and all variants of the NaV1.5 channel, including but not limited to the embryonal isoform, which share significant sequence homology with the canonical NaV 1.5 channel (SEQ ID NO: 1).
  • SEQ ID NO: 1 canonical NaV 1.5 channel
  • the antibodies for use as biomarkers for the diagnosis of Brugada Syndrome in a human being of the invention are directed against a binding site of the extracellular loops of NaV 1.5 channel (SEQ ID NO: 1) and related isoforms.
  • the autoantibodies may be directed also against a binding site exposed in the inner channel protein.
  • the antibodies for use as biomarkers for the diagnosis of Brugada Syndrome of the invention are directed against a binding site of the extracellular loops of NaV 1.5 channel and related isoforms, wherein said binding site may be selected from the group comprising the following sequences: i) VFALIGLQLFMGNLRHKCVRNFTALNGTNGSVEADGLVWESLDLYLS DPENYLLKNGTSDVLLCGNSSDAGTCPEGYRCLKAGENPDHGYTSFDSFA WAFLALFRL (SEQ ID NO:2 ) ii) FGKNYSELRDSDSGLLPRWHMMDFFHAFLIIFRILCG (SEQ ID NO:3) iii) SIMGVNLFAGKFGRCINQTEGDLPLNYTIVNNKSQCESLNLTGELYW TKVKVNFDNVGAGYLALLQ-1414 (SEQ ID NO: 4) iv) VILSIVGTVLSDIIQKYFFSPTLFRVIRLARIGRIL (SEQ ID NO:
  • Said antibodies directed against the NaV 1.5 channel are autoantibodies depicted in a biological sample of a human being affected by Brugada Syndrome.
  • the in vitro method comprises the following steps: a) contacting the biological sample with one or more antigens that specifically bind to the antibodies directed against NaV1.5 channel of (SEQ ID NO:1) and related isoforms; and b) detecting the binding of the antigen to the antibodies in the biological sample.
  • said one or more antigens of step a) bind to the antibodies directed against extracellular loops of NaV1.5 channel of (SEQ ID NO: 1) and related isoforms.
  • said antigens are fragments of at least 5 amino acids, more preferably of at least 7 amino acids from the binding sites of extracellular loops of NaV1.5 channel.
  • said binding sites of extracellular loops of NaV1.5 channel are selected between the sequences SEQ ID NO:2-SEQ ID NO: 14.
  • said antigens are labelled.
  • said antibodies are directed against extracellular loops of NaV1.5 channel of (SEQ ID NO: 1).
  • steroids such as Prednisone, Methylprednisolone, and Dexamethasone
  • medicament like Colchicine and Hydroxychloroquine (Plaquenil) that could reduce the production of autoantibodies may be advantageously used for the treatment of Brugada syndrome.
  • the present invention further contemplates a therapeutic agent specifically targeting the antibodies directed against the NaV 1.5 channel (SEQ ID NO: 1) and related isoforms for use in the treatment of Brugada syndrome.
  • said antibodies are directed against extracellular loops of NaV1.5 channel of (SEQ ID NO: 1) and related isoforms.
  • the therapeutic agent is characterized in that it comprises one or more antigenic fragments of the extracellular loops of the Nav 1.5 channel (SEQ ID NO:1) and related isoforms.
  • the invention further contemplates the use of the antagonist or the autoimmune therapeutic agent in combination with another therapy for Brugada syndrome in order to achieve a more comprehensive and effective treatment.
  • said antigens are labelled or attached to a solid support.
  • said antibodies are labelled with fluorescents.
  • Said solid support is preferably a multi-well plate.
  • said kit is an ELISA kit.
  • the invention is directed to the use of one or more of the autoantibodies against NaV 1.5 channel (SEQ ID NO: 1) and related isoforms for the diagnosis of Brugada Syndrome in a human being by (qualitative or quantitative) detection of the presence thereof in a biological sample, wherein said biological sample is selected from the group consisting of plasma, PBMCs, whole blood, serum and peripheral blood, pericardial fluid, or a combination thereof.
  • said detection step is carried out by using one or more antigens that specifically binds to the autoantibodies against NaV 1.5 channel (SEQ ID NO: 1) and related isoforms.
  • said one or more antigens are selected from the antigenic fragments or epitopes belonging to a binding site of the extracellular loop of NaV 1.5 channel consisting of the sequences SEQ ID NO:2-SEQ ID NO: 14.
  • the invention further relates to an animal model obtained by injecting the plasma from a patient affected by Brugada Syndrome containing autoantibodies being directed against NaV 1.5 channel (SEQ ID NO: 1) and/or related isoforms as above disclosed.
  • said animal model is a rodent, preferably a mouse.
  • a “humanized animal model” 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 mimic human physiological and pathological processes more closely.
  • the humanized animal model is achieved by injecting antigens that target specific antibodies, facilitating the study of the NaV 1.5 channel and its related isoforms in a setting that better represents human biology.
  • FIG. 3 shows representative fluorescence images validating the presence of NaV1.5 autoantibodies in plasma from patients with Brugada syndrome.
  • Panel A Representative fluorescence image displaying BrS positive and negative samples. Red fluorescence highlights NaV 1.5 expression, while green fluorescence indicates the presence of bound human antibodies. The same plasma was tested on both transfected and non-transfected cells (HEK-WT), Panel B) Representative fluorescence image illustrating BrS positive and negative plasma. Red fluorescence indicates NaV1.5 expression, while green fluorescence serves as an indicator of human IgGs.
  • FIG. 5 shows detection of autoantibodies against NaV1.5 protein from plasma of 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. A 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).
  • Co-localization of IgGs from Brugada syndrome patients with and without SCN5A mutations, but not from Post Ablated (PA) patients was studied with anti-rabbit anti-NaV1.5 and anti -human IgG antibodies.
  • FIG. 6 shows detection of autoantibodies against NaV1.5 protein in the plasma of Brugada syndrome patients.
  • Western blot analysis proteins from lysed cells were separated by one-dimensional SDS-PAGE. A 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).
  • - Figure 7 shows a comparison of representative fluorescence image of Brugada syndrome positive and negative plasma.
  • Panel A Representative fluorescence image showing BrS positive and negative plasma. Red fluorescence indicates NaV1.5 expression, while green fluorescence serves as an indicator of human IgGs.
  • Panel B Menders coefficient.
  • - Figure 8 shows effects of autoantibodies from Brugada Syndrome patients on sodium current.
  • Panel A Sodium current profiles in HEK293A cells overexpressing NaV1.5 incubated with either control (CTR, left panel) or Brugada Syndrome serum (BrS, right panel, filled circle).
  • Panel D show families of nifedipine-sensitive calcium current in hiPSC-CMs untreated (left) or incubated with BrS patients plasma (right), showing that the current density is unaffected by the plasma incubation.
  • FIG. 11 shows the identification of putative binding sites of autoantibodies on NaV1.5 protein.
  • Panel A Amino acid sequence ofNaV1.5 channel (SEQ ID NO: 1);
  • Panel B Identification of six potential regions that serve as binding sites for autoantibodies. The 10 amino acids critical for binding on the solid support are highlighted in yellow;
  • Panel C PyMol structure showing the core of the NaV1.5 channel and the extracellular loop.
  • FIG. 12 shows peptide microarray scans of the human plasma of 20 patients with Brugada syndrome (Brl200, Dg2675, Dgl936, Brl211, Dg2821, Dg2533, Dg2429, Dg2388, Dg2830, Dg2655, Dg2677, Dg2828, Brl l96, Dg2720, Dg2818, Dg2819, Brl l45, Br938, Brl204, Brl215) analyzed.
  • EXAMPLE 1 Detection of NaV1.5 channel autoantibodies in plasma from Brugada patients
  • Brugada patients diagnosed with Brugada Syndrome (BrS) at the Arrhythmology Department of IRCCS Policlinico San Donato were included in this study [24], Participants were classified into three main subgroups: 1) BrS patients with SCN5A mutations; 2) BrS patients without SCN5A variants; and 3) healthy controls. Additionally, 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) outcomes were collected from medical records.
  • ICD implantable cardioverter-defibrillator
  • HEK293 cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% glutamine at 37°C under 5% CO2.
  • mice were housed under standard conditions with a 12-hour light/dark cycle and ad libitum access to food and water. The mice were kept in the same controlled environment.
  • Blood (25 ml) was centrifuged at 1000g for 15 minutes to isolate plasma, followed by centrifugation of the supernatant at 2000g for 15 minutes. The supernatant was collected, aliquoted, and stored at -20°C.
  • IgG antibodies were isolated using the PureProteomeTM Protein G Magnetic Bead System. The isolation was performed under non-denaturing conditions with high salt concentration and nearly neutral pH.
  • a full-length cDNA encoding human SCN5A was synthesized and cloned into pcDNA 3.1(+).
  • HEK293 cells were transfected with the pcDNA A(+)ISCN5A construct using ViaFect transfection reagent (Promega) according to the manufacturer's instructions.
  • Cells were growth in medium consisting of Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM glutamine (Merck), and IX penicillin/streptomycin (Euroclone) and selected with G418 at a specified concentration.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • Merck 2 mM glutamine
  • IX penicillin/streptomycin Euroclone
  • the cells were maintained at 37°C in a 5% CO2 and 95% air-humidified atmosphere. Harvested cells were lysed, and the cleared lysate was collected after centrifugation. The successful transfection and expression of NaV1.5 channels were confirmed through Western blot analysis.
  • Cells were lysed in a 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 the BCA assay. Proteins were denatured and reduced in a Laemmli- b-mercaptoethanol mixture at 100°C for 5 min. Subsequently, proteins (30 pg) were loaded onto a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane.
  • HEK293A cells overexpressing NaV1.5 were lysed using RIPA buffer containing a cocktail of protease and phosphatase inhibitors, with subsequent centrifugation at 15,000 rpm for 10 minutes at 4 °C to collect the supernatant. Protein concentrations were determined by the BCA assay (Pierce). Proteins were denatured and reduced with Laemmli buffer containing P-mercaptoethanol (Bio-Rad) and loaded onto a 10% SDS-PAGE gel (Protean Tgx Stain-Free, Bio-Rad) for electrophoresis, followed by transfer to nitrocellulose membranes.
  • the membranes were blocked and probed overnight at 4 °C with the primary anti-NaV1.5 antibody (1 :2000 dilution, Cell Signaling, clone D9J7S), followed by washing and incubation with a secondary anti-rabbit IRDye 800 CW antibody (1 :2000 dilution, LLCOR Biosciences).
  • the membranes were then incubated with BrS patient plasma, either boiled at 100°C for 10 minutes or left untreated, followed by staining with a secondary anti-human IgG-HRP antibody (1 :2000 dilution, Bio-Rad). Bands were visualized using the ECL Advance kit (GE Healthcare) and imaged with the ChemiDoc MP system (Bio-Rad).
  • NaV1.5 channel Immunoprecipitation from protein lysate of cardiac mouse tissue To collect the heart samples, the animals were anesthetized by intraperitoneal injection of medetomidine, 0.5 mg/kg (Orion Pharma S.r.l.) and ketamine, 100 mg/kg (Merial), both diluted in saline. Once the animals were completely unconscious, their thorax was opened, and the heart was perfused with 1 ml KC1 IM to induce diastolic arrest and flushed with 0.9% saline through a cannula inserted into the left ventricle. The heart was then excised, and the left ventricle was separated from the atria and right ventricle.
  • the left ventricle was then divided into 3-mm-thick slices using a specific stainless steel heart matrix (Roboz Surgical Instruments) to create apex, middle, and base specimens.
  • the apex was incubated with 500 ml RIPA Lysis Buffer and homogenized using the tissue homogenizer Lyser® (Qiagen). Each sample was subjected to three homogenization cycles, each lasting 5 minutes at a rate of 25 oscillations per second.
  • the homogenate was kept in ice for 30 minutes and then centrifuged at 10000 ref 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.
  • Immunofluorescence assays were conducted on adult C57BL-6 mouse heart tissue. Left ventricle sections, 12 pm thick, were prepared using a cryostat and mounted onto gelatin-coated histological slides. The sections were thawed, rehydrated, and subjected to antigen unmasking. Non-specific binding was blocked using a solution containing normal donkey serum and bovine serum albumin. Incubation with patient plasma diluted 1 :50 in PBS with 2% NDS and 2% BSA was performed, followed by washing and incubation with an anti-human IgG-FITC secondary antibody.
  • the sections were incubated with a primary rabbit monoclonal anti-NaV1.5 antibody (1 :200 dilution; clone D9J7S, Cell Signaling), followed by washing and incubation with an appropriate anti-rabbit secondary antibody conjugated with Cy3. After further washes, the sections were mounted with Vectashield mounting medium containing DAPI. Images were captured using a Leica Thunder microscope (*40 objective).
  • CBA-IF Cell -based assay immunofluorescence
  • the chip-based automated planar patch-clamp system Patchliner (Nanion Technologies GmbH, Munchen, Germany) was employed to record sodium currents from HEK293A cells transiently transfected with the NaV1.5 channel. After 1 hour incubation at 37°C and 5% CO2 with 5% BrS or Control derived serum, cells were gently tripsinized and resuspended in the extracellular low-sodium recording solution from Nanion (Ref. 08-3004, ionic composition in mM: 80 NaCl, 60 NMDG, 4 KC1, 2 CaCh, 1 MgCh, 5 D-Glucose monohydrate, 10 Hepes; pH 7.4 with HC1, 289 mOsm). Non treated cells were used as internal reference.
  • CsF-based intracellular (Ref. 08-3008, ionic composition in mM: 10 EGTA, 10 Hepes, 10 CsCl, 10 NaCl, 110 CsF; pH 7.2 with CsOH, 280 mOsm) and the extracellular seal enhancer (Ref.
  • the currentvoltage (I-V) relationship and the voltage-dependence of activation of the sodium currents were obtained applying a protocol with incremental 50 ms steps ranging from -80 to +60 mV (holding potential -120 mV).
  • Raw traces recorded by HEK293 A amplifiers were exported with a home-built Python tool and individual traces were analysed using Clampfit 10.7 (Molecular Devices, San Jose, CA, USA), Origin Pro (OriginLab, Norhampton, MA, USA), and GraphPad Prism (GraphPad Software, Boston, MA, USA). Current density was calculated dividing the current amplitude (pA) by the cell capacitance (pF) for each cell.
  • Inward currents were elicited by manual patch-clamp at 37°C in whole-cell configuration applying a step protocol from -80 to 60 mV, 150 ms duration (holding -80 mV).
  • the protocol was applied in absence of any drugs, in presence of 10 pM nifedipine, and in presence of 10 pM nifedipine and 30 pM TTX.
  • Nifedipine sensitive ICaL and TTX-sensitive INa were obtained by subsequent subtraction during the analysis.
  • Intracellular solution was (mM): 135 CsCl, 10 NaCl, 5 EGTA, 2CaC12, 2 TEA-CI, 10 HEPES, 2 MgATP; pH 7.2 with CsOH.
  • NaCl was 80 mM.
  • mice The procedure involving mice was performed according to the animal protocol guidelines described by the Institutional Animal Care and Use Committee (IACUC) authorization no. 425/2022/PR at San Raffaele Scientific Institute (Milan, Italy). Mice C57BL-6 at 50 weeks were maintained ad libitum access to water and standard chow food at room temperature with a 12-h light/dark schedule. They were anesthetized by intraperitoneal injection of medetomidine, 0.5 mg/kg (Orion Pharma S.r.l.) and ketamine, 100 mg/kg (Merial), both diluted in saline solution.
  • IACUC Institutional Animal Care and Use Committee
  • ECG was performed continuously from 10 minutes after induction of anesthesia and 30 minutes after plasma injection using four subcutaneous needle electrodes (stainless steel, 27-gauge, 12 mm length; SEI EMG s.r.l., Cittadella, Italy): two needles were inserted in the forelimbs and one in the left hindlimb, and another needle electrode was placed in the right hindlimb as a ground.
  • a consistent lead configuration without changing the polarity or placement of the subcutaneous needle electrodes before, during and after plasma administration.
  • the leads were placed in the standard Einthoven configuration for mice, ensuring that each mouse underwent the same procedure for accurate results. The lead configuration remained unchanged throughout the plasma infusion challenge.
  • the lead placement for mouse ECG is a general description of the lead placement for mouse ECG:
  • Lead III This lead measures the potential difference between the left forelimb (negative) and the left hindlimb (positive). Electrodes are placed on the left forelimb and the left hindlimb. This is shown in the third line of every ECG experiment.
  • a ground electrode is placed on a neutral area, right hindlimb, to stabilize the signal and reduce noise.
  • Needle electrodes were connected via flexible cables to an amplifier (Micromed, Mogliano Veneto, Italy), then the ECG signal was recorded using System-Plus software (Micromed, Mogliano Veneto, Italy) and sampled at 256 Hz (16 bits) with band-pass filters between 1 and 70 Hz. All animal experiments were performed without prior knowledge of the origin of the plasma samples to ensure the integrity of the results.
  • Mouse NaV1.5 is immunoprecipitated by BrS anti-NaV1.5 autoantibodies
  • Western blot analysis indicate that IgGs from BrS plasma are able to specifically bind NaV1.5 from protein lysate of cardiac mouse tissue ( Figure 2). Magnetic beads coated with IgGs from BrS patients were employed to incubate with mouse cardiac ventricular protein extract. Subsequently, the immunoprecipitated (IP) and immunodepleted (I-) fractions were resolved through Western blot analysis (left blot).
  • mice were assessed using ECG. Continuous ECG monitoring of seven mice expressing the wild-type isoform of NaV1.5 channels that were intravenously administered 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 asystole and death ( Figure 10 A-B).
  • mice receiving control plasma exhibited no ECG alterations, including no ST-segment elevation or conduction disturbances ( Figure 10 C-D).
  • removing autoantibodies from the plasma before infusion prevented the Brugada- like phenotype in mice ( Figure 10 E-F).
  • administering the same BrS plasma with autoantibodies had caused a coved-type ST-segment elevation pattern and a malignant arrhythmic phenotype ( Figure 10 A-B). This evidence supports that the therapeutic approaches and/or medicine capable of reducing autoantibody levels according to the present invention could mitigate the risk of autoantibody-induced arrhythmias.
  • EXAMPLE 2 Identification of Putative NaV1.5 Autoantibody Binding-Sites on NaV1.5 Channel Protein
  • the human NaV1.5 channel has the following amino acid sequence:
  • the resulting NaV 1.5 peptide microarrays contained 327 different peptides printed in duplicate (654 peptide spots) and were framed by additional HA (YPYDVPDYAG SEQ ID NO: 16, 40 spots) and polio (KEVPALTAVETGAT SEQ ID NO: 17, 38 spots) control peptides.
  • microarray was subjected to Rockland blocking buffer MB-070 for 30 min and then pre-stained with the secondary antibody goat anti-human IgG (Fc) DyLight680 (0.1 pg/ml) for 45 min staining in incubation buffer at RT to investigate background interactions that could interfere with the main assays.
  • Fc goat anti-human IgG
  • Quantification of spot intensities and peptide annotation were based on 16-bit gray scale tiff files that exhibit a higher dynamic range than the 24-bit colorized tiff files shown in this report.
  • Microarray image analysis was done with PepSlide® Analyzer a software algorithm breaks down fluorescence intensities of each spot into raw, foreground and background signals, and calculates averaged median foreground intensities. Based on averaged median foreground intensities, intensity maps were generated and interactions in the peptide maps highlighted by an intensity color code with red for high and white for low spot intensities.
  • Innopsys InnoScan 710- IR Microarray Scanner was used with resolution of 20 pm; scanning gains of 50 at low laser power (680 nm, red) and 10 at high laser power (800 nm, green).
  • DII S5-S6 (861-897) 37 aa:
  • AGWDGLLSPILNTGPPYCDPTLPNSNGSRGDCGSPAVGILFFTT (SEQ ID NO: 7)
  • DI-S5-S6 (263-368): SVEADGLVWESLDLYLSDPENYLLKNGTS (SEQ ID NO: 8)
  • DI S5-S6 (263-368): CLKAGENPDHGYTSFDSFAWAFLAL (SEQ ID NOV) Dill S5-S6 (1349-1414): DSDSGLLPRWHMMDFFHAFLII (SEQ ID NO: 10) Dill S5-S6 (1349-1414): VNNKSQCESLNLTGEYWTKVK (SEQ ID NO: 11) DIV S3-S4 (1598-1634): GSGVILSIVGTVLSDIIQKYFFSPT (SEQ ID NO: 12) DIV S5-S6 (1670-1707): MANFAYVKWEAGIDDMFNFQTFANSMLCLF (SEQ ID NO: 13)

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Abstract

La présente invention concerne des auto-anticorps contre le canal NaV1.5 et des isoformes associées, leur utilisation en tant que biomarqueur pour le diagnostic du syndrome de Brugada chez l'être humain et des méthodes associées de détection et d'approche thérapeutique pour cibler cette forme d'auto-immunité nouvellement découverte.
PCT/EP2024/071047 2023-07-24 2024-07-24 Auto-anticorps dirigés contre le canal nav1.5 en tant que biomarqueurs pour le diagnostic du syndrome de brugada Pending WO2025021888A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
EP23187367.0A EP4498088A1 (fr) 2023-07-24 2023-07-24 Autoanticorps dirigés contre la protéine canal nav1.5 comme biomarqueurs pour le diagnostic du syndrome de brugada
EP23187367.0 2023-07-24
US18/387,237 US20250145704A1 (en) 2023-11-06 2023-11-06 AUTOANTIBODIES AGAINST NaV1.5 CHANNEL AS BIOMARKERS FOR THE DIAGNOSIS OF BRUGADA SYNDROME AND RELATED THERAPEUTIC METHODS
US18/387,237 2023-11-06
EP24161814.9A EP4614149A1 (fr) 2024-03-06 2024-03-06 Autoanticorps dirigés contre le canal nav1.5 comme biomarqueurs de diagnostic du syndrome de brugada
EP24161814.9 2024-03-06

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WO2020220136A1 (fr) * 2019-05-02 2020-11-05 The Hospital For Sick Children Détection de biomarqueurs associés au syndrome de brugada
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US20150018251A1 (en) * 2012-02-01 2015-01-15 Centre National De La Recherche Scientifique Protein chips, preparation and use thereof
WO2020220136A1 (fr) * 2019-05-02 2020-11-05 The Hospital For Sick Children Détection de biomarqueurs associés au syndrome de brugada
WO2022180559A1 (fr) * 2021-02-26 2022-09-01 Cardiomix S.R.L. Biomarqueurs sanguins de protéines et de métabolites pour le diagnostic du syndrome de brugada

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