Actinin-related antigenic epitope peptides and uses thereof

Abstract

The present application provides a titin-related antigen epitope peptide and application thereof. The titin antigen epitope peptide of the present application is selected from: (1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 14; and (2) a polypeptide derived from (1) by substitution, deletion or addition of 1-2 amino acids in the amino acid sequence of SEQ ID NO: 14 and retaining the binding ability with titin antibody. The present application first identifies a new titin antigenic peptide segment sequence, which not only expands the existing self-antigen epitope map, but more importantly provides a molecular basis for developing a clinical detection method with higher diagnostic sensitivity and specificity, helps to further improve the pathological mechanism of MG, and provides a new auxiliary diagnostic method and therapeutic target for antibody-negative MG patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to titin-related antigenic epitope peptides and their applications. Background Technology

[0002] Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies targeting the neuromuscular junction. Detecting pathogenic antibodies in peripheral blood is an important auxiliary diagnostic tool. Pathogenic antibodies include, for example, acetylcholine receptor antibodies (AChR-Ab), muscle-specific tyrosine kinase antibodies (MuSK-Ab), low-density lipoprotein-associated protein 4 antibodies (LRP4-Ab), and lanodin receptor antibodies (RyR-Ab). Current clinical diagnosis mainly relies on serological testing of AChR-Ab, MuSK-Ab, LRP4-Ab, and RyR-Ab. Detecting these antibodies, especially in patients with atypical symptoms or in the early stages, can significantly improve diagnostic accuracy.

[0003] Titin antibodies are frequently detected in late-onset myasthenia gravis (MG) and MG patients with thymoma. Titin, a highly elastic microfilamentous protein, is the third most abundant protein in skeletal muscle fibers and plays a crucial role in maintaining muscle structure and function as a molecular spring. Currently, titin antibodies are widely used in the serological auxiliary diagnosis of MG. Regarding the identification of titin autoantigen epitopes, current techniques have identified some key sites, including the classic major immunogenic region located at amino acid positions 13330-13616 (NCBI: NP_596869.4). However, titin has an extremely large molecular weight, containing over 30,000 amino acids, and exhibits complex splicing subtypes. Existing epitopes are insufficient to comprehensively cover all antigenic characteristics of titin. This limitation in understanding directly results in current diagnostic techniques being unable to cover all autoantibodies against titin. Summary of the Invention

[0004] This invention identifies for the first time a novel antigenic peptide sequence of titin, specifically amino acids 26122-26141 (NCBI: NP_596869.4) of human titin, with the amino acid sequence YKKSDDTDWKTSIQSLRGTE (SEQ ID NO: 14). This discovery not only expands the existing autoantigen epitope map but, more importantly, provides a molecular basis for developing clinical detection methods with higher diagnostic sensitivity and specificity. It helps to further elucidate the pathological mechanisms of myocardial infarction (MG) and provides new auxiliary diagnostic tools and therapeutic targets for antibody-negative MG patients.

[0005] In a first aspect, the present invention provides a titin antigenic epitope peptide selected from: (1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 14; and (2) a polypeptide derived from (1) having substituted, deleted or added 1-2 amino acids in the amino acid sequence of SEQ ID NO: 14 and retaining the ability to bind to titin antibodies.

[0006] In some embodiments, the titin may be titin from mammals (e.g., humans, rats, mice).

[0007] In a second aspect, the present invention provides a fusion protein comprising the titin antigen epitope peptide described in the first aspect and an optional tag sequence.

[0008] In some embodiments, the tag sequence includes, but is not limited to, GST tag sequence, FLAG tag sequence, His tag sequence, MYC tag sequence, fluorescent tag sequence (e.g., green fluorescent sequence, orange fluorescent sequence, red fluorescent sequence, and yellow fluorescent sequence, such as mCherry), etc.

[0009] In a third aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to the titin antigenic epitope peptide described in the first aspect or the fusion protein described in the second aspect, and the antibody or the antigen-binding fragment thereof is capable of specifically binding to titin.

[0010] In some embodiments, the antibody includes a monoclonal antibody, a polyclonal antibody, or an antiserum, and the antigen-binding fragment includes Fab, Fab', F(ab')2, a single-chain antibody (scFv), a dimerized V region (biantibody), or a disulfide-stabilized V region.

[0011] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6.

[0012] In some embodiments, the heavy chain variable region (VH) of the antibody or its antigen-binding fragment may contain an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 7; and the light chain variable region (VL) of the antibody or its antigen-binding fragment may contain an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 9.

[0013] In some embodiments, the antibody or its antigen-binding fragment may comprise a heavy chain (H) and a light chain (L), wherein the heavy chain may comprise an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 8; and the light chain may comprise an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 10.

[0014] In some embodiments, the antibody is a monoclonal antibody.

[0015] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (biantibody), and disulfide bond-stabilized V region (dsFv).

[0016] In a fourth aspect, the present invention provides an isolated polynucleotide encoding the titin antigen epitope peptide described in the first aspect, the fusion protein described in the second aspect, or the antibody described in the third aspect.

[0017] In a fifth aspect, the present invention provides an expression vector comprising the polynucleotides described in the fourth aspect.

[0018] In a sixth aspect, the present invention provides a host cell containing the expression vector described in the fifth aspect, or integrating the polynucleotides described in the fourth aspect into its genome.

[0019] In some embodiments, the cells are selected from prokaryotic and eukaryotic cells, preferably eukaryotic cells, and more preferably mammalian cells. Examples of the cells in some embodiments include, but are not limited to, HEK293T cells, HeLa cells, and Hep2 cells.

[0020] In a seventh aspect, the present invention provides an ELISA kit for detecting titin antibodies (particularly, autoantibodies), the ELISA kit comprising: the titin antigen epitope peptide described in the first aspect or the fusion protein described in the second aspect; and / or the antibody described in the third aspect as a positive standard.

[0021] The ELISA kit may also include common laboratory devices or reagents known in the art for assembling ELISA kits, such as microplates, TMB chromogenic solution, enzyme-labeled secondary antibodies, etc.

[0022] In some embodiments, the ELISA kit of the present invention includes the titin antigen epitope peptide described in the first aspect and the antibody described in the third aspect.

[0023] In some embodiments, the ELISA kit of the present invention can be used as an adjunct to the diagnosis of myasthenia gravis.

[0024] In an eighth aspect, the present invention provides a CBA kit for detecting titin antibodies (particularly autoantibodies), the kit comprising: the antibody described in the third aspect, the polynucleotide described in the fourth aspect, the expression vector described in the fifth aspect, or the host cell described in the sixth aspect.

[0025] The CBA kit may also include common laboratory devices or reagents known in the art for assembling CBA kits, such as cell culture plates (well plates), cell fixatives, permeabilization solutions, blocking solutions, DAPI staining solutions, fluorescently labeled secondary antibodies (e.g., Alexa Fluor 488 / 594 labeled goat anti-human IgG), phosphate buffer, and kit instructions.

[0026] In some embodiments, the CBA kit of the present invention can be used for the auxiliary diagnosis of myasthenia gravis. Attached Figure Description

[0027] Figure 1 The results of the cell-based indirect immunofluorescence assay shown in Example 5 are illustrated. Figure A shows the staining results of normal human IgG (negative control) in HeLa cells expressing a new titin epitope peptide; Figure B shows the staining results of the human anti-titin antibody obtained in Example 2 (positive control) in HeLa cells expressing a new titin epitope; Figure C shows the staining results of serum from patients with myasthenia gravis (experimental group) in HeLa cells expressing a new titin epitope. Scale bar: 200 μm.

[0028] Figure 2The absorbance results of the binding specificity test between human anti-titin antibody and three peptides (His, w / o motif, and w motif) in the enzyme-linked immunosorbent assay (ELISA) of Example 6 are shown. His is a short peptide sequence (HHHHHH) consisting of six consecutive histidine residues, serving as a negative control to measure the background signal of the detection system. w / o motif is a negative control peptide without the antigen recognition epitope, and w motif is the experimental group corresponding to the antigen epitope peptide containing the antigen recognition epitope. This indicates a statistically significant / extremely significant difference between groups (P<0.0001).

[0029] Figure 3 The absorbance results of the reaction between serum and the neoantigen epitope of titin in the myasthenia gravis validation cohort of Example 7 are shown. The Healthy group consisted of serum from 118 healthy individuals, and the Patients group consisted of serum from 187 myasthenia gravis patients. This indicates a statistically significant / extremely significant difference between groups (P<0.0001).

[0030] Figure 4 The results of the positive percentage of titin neoantigen epitope recognition antibodies in the myasthenia gravis validation cohort in Example 7 are shown. Figure A shows the positive rate of titin neoantigen epitope peptide recognition antibodies in the cohort of 187 myasthenia gravis patients, which was 19.8%. Figure B shows the positive rate of titin neoantigen epitope recognition antibodies in 160 serologically positive patients in this cohort who tested positive for anti-acetylcholine receptor (AChR), muscle-specific kinase (MuSK), and low-density lipoprotein receptor-associated protein 4 (LRP4) antibodies, which was 20.0%. Figure C shows the positive rate of titin neoantigen epitope peptide recognition antibodies in 27 serologically negative patients in this cohort who tested negative for the aforementioned AChR, MuSK, and LRP4 antibodies, which was 18.5%.

[0031] Figure 5 Immunostaining results of muscle tissue from normal human IgG (A) and human anti-tipin antibody (B) in Example 8. Scale bar: 50 μm. Detailed Implementation

[0032] The present invention will be described in detail below by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] Reagents: MACS tissue preservation solution, purchased from Miltenyi Biotec, catalog number: 130-100-008; RPMI 1640 medium, purchased from Gibco, catalog number: 11875093; Collagenase II, purchased from Sigma, product number: C2-28-100MG; DNase I, purchased from Roche, item number: 10104159001; Trypsin, purchased from Sigma, catalog number: T4799-5G; Red blood cell lysis buffer (RBC Lysis Buffer), purchased from BioLegend, catalog number: 420301; AO / PI fluorescent dye, purchased from Logos Biosystems, catalog number: F23001; Phosphate-buffered saline (PBS), purchased from Zhongshan Jinqiao, catalog number: ZLI-9061; Fetal bovine serum (FBS), purchased from Gibco, catalog number: 10099141C; High glucose complete culture medium (DMEM) was purchased from Pronosai, catalog number: PM150210B; The transfection reagent, Lipofectamine 2000, was purchased from Invitrogen, catalog number: 11668019. Paraformaldehyde, purchased from Sigma, item number: P6148-5KG; Triton X-100, purchased from Sigma, part number: T8787; Normal human immunoglobulin G (hIgG), purchased from Merck, catalog number I4506; Alexa Fluor 488 Anti-human IgG secondary antibody, purchased from Thermo Fisher Scientific, catalog number: A-11013; Mounting Medium With DAPI, purchased from Abcam, item number: ab104139; Pierce® streptavidin-coated 96-well plates, purchased from Thermo Fisher Scientific, item number: 15121; Bovine serum albumin (BSA), purchased from Sigma, product number: V900933; HRP-labeled secondary antibody, purchased from Abcam, catalog number: ab6759; Two-component TMB colorimetric kit, purchased from ProteinTech, catalog number: PK10004.

[0036] instrument: Pure water system: Millipore Direct-Q® 5 UV Water Purification System; Pipettes: Eppendorf; Upright fluorescence microscope: Nikon ECLIPSE Ni-U; BioTek Synergy H1: Multifunctional Microplate Analyzer

[0037] Example 1: Obtaining paired human antibody sequences by BCR sequencing 1. Patient Enrollment: Patients with positive acetylcholine receptor antibodies and a clinically confirmed diagnosis of myocardial infarction (MG) were included, and thymus tissue was obtained from them. Patients had signed informed consent.

[0038] 2. Thymus Tissue Processing to Obtain Single-Cell Suspension: Fresh human thymus tissue was transferred to sterile gauze to remove residual blood, cut into small pieces, and immersed in MACS tissue preservation solution. The tissue was then thoroughly minced with scissors and placed in a digestion solution containing collagenase II (0.2%) and DNase I (0.1 mg / ml), and digested at 37°C for 20 minutes. Trypsin was then added for an additional 8 minutes of digestion. The tissue suspension was homogenized by pipetting and filtered through a 40-micron Falcon™ cell filter. The filtrate was centrifuged at 500g for 5 minutes to collect the cell pellet, and the supernatant was discarded. The cell pellet was resuspended in erythrocyte lysis buffer to lyse the erythrocytes. After 5 minutes, RPMI 1640 medium was added, and the mixture was centrifuged at 500g for 5 minutes to remove the supernatant. Finally, the cells were washed with RPMI 1640 and centrifuged at 200g to obtain a single-cell suspension. Cell viability and concentration were detected using a fluorescence cell counter after staining with AO / PI fluorescent dye.

[0039] 3. 10X Genomics Transcriptome Sequencing, VDJ 5′ RACE Amplification Sequencing, and Data Analysis: Using the 10X Genomics Chromium™ microfluidic system, thymic cells (with dead cells removed) were reacted with barcode-labeled gel beads in a water-in-oil single-cell reaction microsystem to construct cDNA libraries. The droplet-encapsulated reverse-transcribed cDNA was divided into two aliquots and used the Chromium Single-Cell 5′ Library Kit (10x Genomics, PN-1000190) and the Chromium Single-Cell V(D)J Amplification Kit (10x Genomics, PN-1000005) for 5′ single-cell transcriptome and B-cell VDJ sequencing library construction, respectively. Sequencing was performed on the Illumina NovaSeq platform. The collected 5′ single-cell transcriptomes were sequenced on an Illumina NextSeq 500. The obtained FASTQ file was first input into the Linux-based CellRanger software for sequencing data cleaning. Then, it was compared with the reference genome of CellRanger GRCh38 based on the GEX sequencing library. A single-cell gene expression matrix was generated based on the cell-specific barcode. Memory B cells / plasma cells expressing mature BCR / antibodies were identified through single-cell transcriptome analysis. Based on the obtained single-cell transcriptome library data, the usage of paired heavy / light chain variable regions V, (D), J and constant C region gene families was analyzed. BCR sequences using the same variable regions V (D) and J for heavy and light chains and with sequence similarity greater than 85% were defined as the same clone.

[0040] Example 2: Expression of human anti-tipin antibody 1. Monoclonal antibodies (mAbs) were prepared based on the single-cell sequencing data from Example 1. Light and heavy chain sequences of clonally expanded B-cell receptors (BCRs) were obtained from the single-cell sequencing data. The selected VH and VL sequences, after codon optimization (the codon-optimized nucleotide sequences of VH and VL correspond to SEQ ID NO: 11 and SEQ ID NO: 12, respectively), were inserted into a eukaryotic expression plasmid (PTT5 backbone from the Genentech plasmid library) containing the human IgG1 Fc domain (SEQ ID NO: 13), resulting in light chain and heavy chain expression plasmids, respectively. The antibodies were expressed in vitro in GUeasyCHO cells (purchased from Genentech).

[0041] 2. Heavy chain expression plasmids and light chain expression plasmids were transfected into GUeasyCHO cells by electroporation.

[0042] 3. Collect the supernatant 5-6 days after transfection, centrifuge, filter, and purify using Protein A magnetic beads (Zhihui Life Science, SM003100).

[0043] 4. The purified protein was desalted, concentrated, and its purity was assessed by SDS-PAGE electrophoresis and FastStain staining. Candidate monoclonal antibodies were obtained.

[0044] The amino acid sequence of the heavy chain (H) of the anti-tipin antibody of the present invention is shown in SEQ ID NO: 8; the amino acid sequence of the light chain (L) is shown in SEQ ID NO: 10. Using bioinformatics analysis of the sequencing results, and after IMGT IgBlast alignment, the amino acid sequence of the variable region (VH) of the heavy chain (SEQ ID NO: 7) and the amino acid sequence of the variable region (VL) of the light chain (SEQ ID NO: 9) were obtained.

[0045] IMGT IgBlast analysis revealed that the heavy chain variable region contains HCDR1 with the amino acid sequence GNFTEA (SEQ ID NO: 1), HCDR2 with the amino acid sequence KSKTDGET (SEQ ID NO: 2), and HCDR3 with the amino acid sequence CMVDLDFW (SEQ ID NO: 3). The light chain variable region contains LCDR1 with the amino acid sequence RASQGINNFLA (SEQ ID NO: 4), LCDR2 with the amino acid sequence GASTLQS (SEQ ID NO: 5), and LCDR3 with the amino acid sequence CQQYTIYPWTF (SEQ ID NO: 6).

[0046] Example 3: Phage immunoprecipitation sequencing for identification of anti-tipin antibodies and neotipin epitope sequences Based on the NCBI / UniProt protein database, this study designed a peptide library covering the full length of human proteins. This library contains 590,000 peptides, including the human titanin described in this invention. Each peptide is 56 amino acids long, with a 50% overlap between adjacent peptides. A T7 phage display library was constructed using high-throughput DNA synthesis technology to express this peptide library. After co-incubating the monoclonal antibody from Example 2 with the phage display peptide library, the antibody-antigen (phage) complex was enriched using immunoprecipitation. Antibody-bound phages were captured using Protein G / A magnetic beads, and the phage DNA was subsequently amplified by PCR, library constructed, and subjected to next-generation sequencing (NGS) to obtain the set of peptide sequences recognized by the antibody in the sample. Integration analysis of these sequences revealed that the protein antigen targeted by the monoclonal antibody is amino acids 26122-26141 of human titanin, with the amino acid sequence YKKSDDTDWKTSIQSLRGTE (SEQ ID NO: 14).

[0047] Example 4: Synthesis of antigenic peptides using solid-phase polypeptide synthesis method (SPSS) In this embodiment, SPSS was used to prepare the antigen peptide (performed by GenScript Biotech). In short, SPSS involves sequentially coupling amino acids onto a resin to form a peptide chain. After sequence synthesis, the N-terminal Fmoc protecting group is first deprotected, followed by the deprotection of the side chain protecting groups. The peptide is then cleaved from the resin to obtain the antigen peptide product.

[0048] Example 5: Detection of the interaction between anti-tipin antibody and neoantigen epitope peptide of titin based on cell immunofluorescence assay In this embodiment, the interaction between the anti-tipin antibody and the neoantigen epitope peptide of titin was confirmed using cell-based immunofluorescence assay (CBA). In the CBA method, the antigenic protein expressed in cells retains its native spatial structure after fixation, exhibiting better antigenicity. The CBA method has higher sensitivity. The specific method is as follows: 1. HeLa cells (Neuroimmunology Center, Xuanwu Hospital) were cultured in 10% FBS + DMEM high-glucose medium at 37°C in a 5% CO2 incubator.

[0049] 2. When the cell density reaches 40%-50%, the optimized codon coding sequence of the titin antigen epitope peptide, YKKSDDTDWKTSIQSLRGTE (SEQ ID NO:14), along with the mCherry fluorescent tag (SEQ ID NO:16) and the flexible sequence T2A (SEQ ID NO:17), is inserted into the pcDNA3.1(+) eukaryotic expression plasmid vector (Thermo Fisher Scientific, V79020). The resulting titin sequence recombinant plasmid vector pcDNA3.1-mCherry-T2A-Titin is transfected into cells using the transfection reagent Lipofectamine 2000. Fresh culture medium is replaced 6 hours after transfection.

[0050] 3. Cell fixation: 24-48 hours after transfection, when the cell density reaches 80%-90% and mCherry fluorescence is visible under a microscope, discard the culture medium, fix the cells with 4% paraformaldehyde at room temperature for 5-15 minutes, and wash 3 times with PBS.

[0051] 4. Cell permeation blocking: Using permeation buffer (PBS solution containing 0.3% Triton X-100), cells were permeated at room temperature for 5-15 minutes, followed by washing with PBS 3 times. Cells were then treated with blocking buffer (PBS buffer containing 5% BSA) at room temperature for 30 minutes to block non-specific binding sites, followed by washing with PBS 3 times.

[0052] 5. Dilute the anti-titin antibody obtained in Example 2 (using normal human immunoglobulin G (hIgG) as a control) in 200 µL PBS to a final concentration of 5 μg / mL, and add it to the cells to be tested.

[0053] 6. Incubate at 37°C in the dark for 1 hour, then wash 5 times with PBS.

[0054] 7. Add Alexa Fluor 488 Anti-human IgG secondary antibody diluted 1:1000 in PBS and incubate for 30 minutes; wash 5 times with PBS, mount with Mounting Medium With DAPI, and observe the red and green fluorescence signals under a 20X fluorescence microscope objective.

[0055] The results are as follows Figure 1 As shown. From Figure 1 It can be seen that the human anti-tipin antibody in this invention recognizes the neoantigen epitope peptide of titin, and there are autoimmune antibodies that recognize the neoantigen epitope peptide in the serum of patients with myasthenia gravis, proving that this invention can be used to develop a CBA detection kit for detecting titin antibodies.

[0056] Example 6: Enzyme-linked immunosorbent assay (ELISA) to detect the interaction between anti-tipin antibody and neoantigen epitope peptide of titin. In this embodiment, the interaction between the anti-titin antibody obtained in Example 2 of the present invention and the neoantigen epitope peptide of titin was confirmed by enzyme-linked immunosorbent assay (ELISA).

[0057] ELISA detection was performed using Pierce® streptavidin-coated 96-well plates and a two-component TMB colorimetric kit.

[0058] The steps are as follows: 1. Coat the 96-well plate with Pierce® streptavidin and equilibrate at room temperature for 30 minutes; 2. Wash three times (200 µL / well) with washing buffer (25 mM Tris, 150 mM NaCl, pH 7.2, containing 0.1% BSA and 0.05% Tween-20). 3. Add 100 µL of biotinylated titanin neoantigen epitope peptide (w motif, YKKSDDTDWKTSIQSLRGTE, dissolved in wash buffer, final concentration 10 µg / mL) to each well; or biotinylated His protein (His, HHHHHH (SEQ ID NO:18), dissolved in wash buffer, final concentration 10 µg / mL) as a negative control for the background signal of the test detection system; or biotinylated titanin peptide without neoantigen recognition epitope as a negative control (w / o motif, GAPITGYTVEYKKSDDTDWK (SEQ ID NO:19), dissolved in wash buffer, final concentration 10 µg / mL).

[0059] 4. Incubate at room temperature with shaking for 1 hour, wash 3 times with the washing buffer from step 2, add 100µL of anti-tipin antibody working solution (anti-tipin antibody from Example 2 diluted 1:10 with washing buffer), and incubate at room temperature with shaking for 1 hour. 5. Wash again, add 100µL of HRP-labeled secondary antibody (diluted according to the manufacturer's recommended ratio), and incubate at room temperature with shaking for 30 minutes; 6. After washing thoroughly 5 times, add 100µL of TMB substrate and develop the color in the dark for 15-30 minutes; 7. Terminate the reaction (100µL 2M H2SO4), and measure the absorbance at a wavelength of 450nm.

[0060] The absorbance results of His protein, negative control titin peptide, titin neoantigen epitope peptide, and anti-titin antibody were quantitatively analyzed to obtain... Figure 2 .

[0061] from Figure 2 As can be seen, the anti-titin antibody obtained in Example 2 showed only a weak absorbance (Absorbance 450nm) against the negative control His protein, demonstrating that the detection system has a good background signal. w / o motif is the non-recognition epitope peptide in biotinylated titin, and w motif is the neoantigen epitope sequence of biotinylated titin. The experimental results showed that the antibody only had a strong antigen-antibody reaction with w motif, which proves the specificity of the reaction between the antibody and the antigen in this invention, as well as the accuracy and sensitivity of this enzyme-linked immunosorbent assay detection system.

[0062] Example 7: Enzyme-linked immunosorbent assay (ELISA) to detect the interaction between patient serum and titin neoantigen epitope peptide. In this embodiment, the interaction between serum from MG patients and the neoantigen epitope peptide of titin was confirmed by enzyme-linked immunosorbent assay (ELISA). MG patients: Myasthenia gravis patients diagnosed by the Department of Neurology, Xuanwu Hospital, from the sample bank of the Neuroimmunology Center of Xuanwu Hospital. Healthy individuals: Individuals with no relevant medical history, normal physical examination indicators, and negative AChR-Ab in the sample bank of the Neuroimmunology Center of Xuanwu Hospital, excluding those who have been infected / treated with immunotherapy within the past 3 months and those who are pregnant / lactating.

[0063] ELISA detection was performed using Pierce® streptavidin-coated 96-well plates and a two-component TMB colorimetric kit.

[0064] The steps are as follows: 1. Coat the 96-well plate with Pierce® streptavidin and equilibrate at room temperature for 30 minutes; 2. Wash three times (200 µL / well) with washing buffer (25 mM Tris, 150 mM NaCl, pH 7.2, containing 0.1% BSA and 0.05% Tween-20). 3. Add 100 µL of biotinylated titin neoantigen epitope peptide (w motif, YKKSDDTDWKTSIQSLRGTE, dissolved in wash buffer, final concentration 10 µg / mL) to each well. 4. Incubate at room temperature with shaking for 1 hour, wash 3 times with the washing buffer from step 2, add 100µL of the sample to be tested (serum from healthy individuals or patients with myasthenia gravis diluted 1:10 with washing buffer), and incubate at room temperature with shaking for 1 hour. 5. Wash again, add 100µL of HRP-labeled secondary antibody (diluted according to the manufacturer's recommended ratio), and incubate at room temperature with shaking for 30 minutes; 6. After washing thoroughly 5 times, add 100µL of TMB substrate and develop the color in the dark for 15-30 minutes; 7. Terminate the reaction (100µL 2 M H2SO4), and measure the absorbance at a wavelength of 450nm.

[0065] Figure 3 The absorbance results of the serum reaction with the neoantigen epitope of titin in the myasthenia gravis validation cohort of Example 7 are shown. From Figure 3 It can be seen that the absorbance (absorbance 450nm) of the patient group was significantly higher than that of the healthy group, and the difference was statistically significant. This indicates that the neoantigen epitope of myosin discovered in this invention can specifically bind to autoantibodies in the serum of patients with myasthenia gravis, and can effectively distinguish patients with myasthenia gravis from healthy individuals.

[0066] The absorbance results of the neoantigen epitope peptide of titin and the serum of MG patients were quantitatively analyzed to obtain... Figure 4 Figure A is used as a reference, and Figures B and C are obtained by quantitative statistical analysis of the patients' clinical serological results. This invention tested the serum of 187 MG patients and 118 healthy individuals. The 95th percentile of serum absorbance in the healthy individuals was set as the positive cutoff value. Patient serum absorbance exceeding the cutoff value was considered positive for anti-connectin neoantigen epitope recognition antibody. Figure 4 (Medium yellow), the patient's serum absorbance is below the threshold value, indicating a negative result (…). Figure 4 (Medium blue).

[0067] from Figure 4 It can be seen that the novel titin antigen epitope peptide discovered in this invention can bind to autoantibodies in the serum of MG patients. Its positive detection rate of 19.8% suggests that the epitope peptide has the potential to be converted into a serological detection reagent, providing a new target for the auxiliary diagnosis of MG and expanding the existing serological detection system based on classic antigens such as acetylcholine receptor (AChR) and muscle-specific tyrosine kinase (MuSK).

[0068] Example 8: Tissue-based immunofluorescence detection In this embodiment, rat muscle tissue was further used to test whether the human anti-tipin antibody of the present invention could recognize rat-derived native titin. The specific experimental steps are as follows: 1. Frozen rat muscle tissue 30μm sections (male SD rats, 180-200g purchased from Vital River, prepared into sections by tissue perfusion, PFA fixation, dehydration, embedding, etc., and 30μm thick sections were made using a Leica microtome) were taken out of the -80℃ freezer and thawed. 2. Wash the rewarmed sections obtained in step 1 with PBS for 5 minutes; 3. Add the washed sections from step 2 to 50 μL of PBS solution containing 0.3% Triton X-100 for 20 minutes to perforate the tissue membrane; 4. After the membrane-breaking treatment in step 3, add 50 μL of 5% BSA dissolved in PBS to the tissue for 1 hour to block the tissue; 5. Incubate the blocked sections from step 4 with 50 μL of purified anti-tipin antibody obtained in Example 2 or negative control normal human IgG diluted in PBS at 2 μg / mL at 4°C overnight; 6. Wash the incubated sections from step 5 three times with PBS; 7. Incubate the washed sections from step 6 with the anti-human fluorescent secondary antibody AlexaFluor 488 Anti-human IgG diluted 1:2000 in PBS at room temperature for 1 hour; 8. Wash the incubated sections from step 7 three times with PBS; 9. Add DAPI-containing mounting medium to the washed slices from step 8, and air dry at room temperature for storage; 10. Observe the section obtained in step 9 under a 10X fluorescence microscope and take fluorescence photographs.

[0069] Test results as follows Figure 5 As shown, the human anti-tipin antibody reacted with rat muscle tissue, displaying alternating light and dark striped signals, and titin staining was visible in the rat muscle. Since titin is a cytoskeletal protein in skeletal muscle sarcomeres, it exhibits an alternating light and dark striped distribution after immunofluorescence staining, which is a typical structural feature of sarcomeres. This indicates that the human anti-tipin monoclonal antibody of this invention can recognize titin in rat tissue on rat muscle tissue sections.

[0070] sequence list HCDR1: GNFFTEA (SEQ ID NO: 1) HCDR2: KSKTDGET (SEQ ID NO: 2) HCDR3: CMVDLDFW (SEQ ID NO: 3) VH: EVRLVESGGGLVKPRGSLRLSCAGSGFNFTEAWITWVRQAPGKGLEWVGLMKSKTDGETTHFPAAMKDRSSLSRDDSRKTAYLHMSDLKVDDTAVYFCMVDLDFWGPGTLVTVSS(SEQ ID NO: 7) H: MHSSALLCCLVLLTGVRAEVRLVESGGGLVKPRGSLRLSCAGSGFNFTEAWITWVRQAPGKGLEWVGLMKSKTDGETTHFPAAMKDRSSLSRDDSRKTAYLHMSDLKVDDTAVYFCMVDLDFWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 8) LCDR1: RASQGINNFLA(SEQ ID NO: 4) LCDR2: GASTLQS(SEQ ID NO: 5) LCDR3: CQQYTIYPWTF(SEQ ID NO: 6) VL: DVQMTQSPSSLSASVGDRVTITCRASQGINNFLAWFQQKPGKAPKSLIYGASTLQSGVPSRFSGSGSGTDFSLTISSLQPDDFATYYCQQYTIYPWTFGQGTNVEIK(SEQ ID NO: 9) L: MHSSALLCCLVLLTGVRADVQMTQSPSSSLSASVGDRVTITCRASQGINNFLAWFQQKPGKAPKSLIYGASTLQSGVPSRFSGSGSGTDFSLTISSLQPDDFATYYCQQYTIYPWTFGQ GTNVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:10) The nucleotide sequence after codon optimization of the amino acid sequence of SEQ ID NO:7: GAAGTGCGGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCTAGAGGCAGCCTGAGACTGAGCTGTGCCGGCAGCGGCTTCAATTTCACAGAGGCCTGGATCACCTGGGTCCGACAGGCTCCTGGAAAAGGCCTGGAATGGGTCGGACTGATGAAGTCCAAGACCGACGGCGAG ACAACCCACTTTCCTGCCGCCATGAAGGACAGAAGCAGCCTGTCCAGAGATGACAGCAGAAAGACCGCCTACCTGCACATGAGCGACCTGAAGGTGGACGATACCGCCGTGTACTTCTGTATGGTGGACCTGGACTTTTGGGGCCCTGGCACACTGGTCACAGTGTCTAGT (SEQ ID NO:11) The nucleotide sequence after codon optimization of the amino acid sequence of SEQ ID NO:9: GACGTGCAGATGACACAGAGCCCTTCTAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACATGTAGAGCCAGCCAGGGGATCAACAACTTCCTGGCCTGGTTCCAGCAGAAGCCTGGCAAGGCCCCTAAGAGCCTGATCTACGGCGCTAGCACACTGCAGAGCGGAGTGCCTAGCAGATTTTCTGGCAGCGGCTCCGGCACCGATTTCAGCCTGACAATCAGCAGCCTGCAGCCTGACGACTTCGCCACCTACTACTGCCAGCAGTACACAATCTACCCCTGGACCTTTGGCCAGGGCACCAACGTGGAAATCAAG (SEQ ID NO:12) Sequence structure of Human IgG1: GCCTCTACAAAGGGCCCTAGTGTGTTCCCTCTGGCTCCCAGCAGCAAGTCTACATCTGGCGGAACAGCCGCTCTGGGCTGCCTGGTCAAGGATTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCACTGACAAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAAAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACAGTGCCTAGCTCTAGCCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTTTACCCCAGCGACATTGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAGTGA(SEQ IDNO: 13) Titin-related antigenic epitope peptide: YKKSDDTDWKTSIQSLRGTE (SEQ ID NO: 14) SEQ ID NO: 14 antigenic epitope peptide optimized codon-encoding sequence: TACAAGAAGAGCGACGACACCGACTGGAAGACCAGCATCCAGAGCCTGAGAGGCACAGAA (SEQ ID NO: 15) mCherry fluorescent tag encoding sequence: ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAG (SEQ ID NO: 16) Flexible sequence T2A: GGTGGCGGTTCTGGC (SEQ ID NO:17) His: HHHHHH (SEQ ID NO:18) Negative control titin peptide (w / o motif): GAPITGYTVEYKKSDDTDWK (SEQ ID NO:19).

Claims

1. A titin antigenic epitope peptide, said antigenic epitope peptide being a polypeptide with the amino acid sequence shown in SEQ ID NO:

14.

2. A fusion protein comprising the titin antigen epitope peptide as described in claim 1 and an optional tag sequence.

3. An antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and The light chain variable region includes LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO:

6.

4. The antibody or its antigen-binding fragment according to claim 3, wherein: The heavy chain variable region of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 85% identity with SEQ ID NO: 7; and the light chain variable region of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 85% identity with SEQ ID NO:

9.

5. The antibody or antigen-binding fragment thereof according to claim 3, wherein, The antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 85% identity with SEQ ID NO: 8; and the light chain comprising an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 85% identity with SEQ ID NO:

10.

6. The antibody or antigen-binding fragment thereof according to any one of claims 3-5, wherein, The antibody is a monoclonal antibody; The antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody, dimerized V region, and disulfide bond-stabilized V region.

7. An isolated polynucleotide encoding the titin antigenic epitope peptide as claimed in claim 1, or the fusion protein as claimed in claim 2, or the antibody or antigen-binding fragment thereof as claimed in any one of claims 3 to 6.

8. An expression vector comprising the polynucleotide as described in claim 7.

9. A host cell containing the expression vector as described in claim 8, or having the polynucleotide as described in claim 7 integrated into its genome.

10. An ELISA kit for detecting titin antibodies, the kit comprising: The myosin antigen epitope peptide as described in claim 1 or the fusion protein as described in claim 2; and / or The antibody or its antigen-binding fragment as described in any one of claims 3 to 6 is used as a positive standard.

11. The ELISA kit for detecting titin antibodies according to claim 10, wherein, The ELISA kit is used as an adjunct to the diagnosis of myasthenia gravis.

12. A CBA kit for detecting titin antibodies, the kit comprising: The antibody or its antigen-binding fragment as described in any one of claims 3 to 6.

13. The CBA kit for detecting titin antibodies according to claim 12, wherein, The CBA kit is used as an adjunct to the diagnosis of myasthenia gravis.

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