Antibodies against the neuronal intranuclear inclusion disease pathogenic protein polygn2c-iso2, methods of making and uses thereof

Abstract

The application discloses antibodies for neuron intranuclear inclusion disease pathogenic protein PolyGN2C-iso2 and a preparation method and application thereof. The antibody or antigen binding fragment thereof can specifically bind to PolyGN2C-iso2, and comprises heavy chain CDR1-3 shown in SEQ ID NO. 1-3 and light chain CDR1-3 shown in SEQ ID NO. 4-6. The antibody of the application has extremely low cross-reaction with uN2CpolyG, has extremely high accuracy, and thus helps to realize accurate diagnosis of neuron intranuclear inclusion disease. In addition, the monoclonal antibody of the application can treat neuron intranuclear inclusion disease caused by PolyGN2C-iso2.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology, and in particular relates to an antibody targeting the neuronal intranuclear inclusion body disease pathogenic protein PolyGN2C-iso2, its preparation method, and its application. Background Technology

[0002] Intranuclear inclusion body disease (NIID) is a rare, progressive neurodegenerative disease with diverse clinical manifestations. Its pathogenesis remains unclear, and there is currently no effective treatment. Current research indicates that the pathogenesis of NIID is closely related to the abnormal amplification of the GGC trinucleotide repeat in the NOTCH2NLC gene. Current research mainly focuses on a polyglycine (polyG) protein called uN2CpolyG, translated from transcript variant 1 of the NOTCH2NLC gene. The academic community generally believes that the abnormal accumulation and aggregation of uN2CpolyG protein is the core cause of neuronal damage and dysfunction.

[0003] One of the clinical auxiliary diagnostic methods for NIID is the detection of p62-positive intranuclear inclusion bodies in skin sweat gland cells or nerve tissue. However, p62 is a general marker related to autophagy and lacks specificity for NIID. For example, p62 histochemical staining cannot distinguish between NIID disease and fragile X-related tremor / ataxia syndrome (FXTAS). Histopathological staining of p62 also cannot distinguish the specific pathogenic protein and cannot serve as a precise therapeutic target. Summary of the Invention

[0004] To address at least some of the technical problems existing in the prior art, this invention is the first to identify a novel pathogenic protein, PolyGN2C-iso2, produced by NOTCH2NLC transcript 2 in NIID patients and confirm its independent pathogenicity. Specifically, this invention includes the following:

[0005] In a first aspect, the present invention provides an isolated neoantigen peptide, which is the neuronal intranuclear inclusion body disease pathogenic protein PolyGN2C-iso2, said neoantigen peptide having the amino acid sequence shown in SEQ ID NO.12.

[0006] In a second aspect, the present invention provides an antibody or an antigen-binding fragment thereof capable of specifically binding to the neuronal intranuclear inclusion body pathogenic protein PolyGN2C-iso2, said antibody or antigen-binding fragment thereof comprising the heavy chain CDR1-3 shown in SEQ ID NO. 1-3 and / or the light chain CDR1-3 shown in SEQ ID NO. 4-6.

[0007] In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention has any one of the amino acid sequences shown in (I)-(III): (I) The heavy chain variable region sequence shown in SEQ ID NO.7 and / or the light chain variable region sequence shown in SEQ ID NO.8; (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function; (III) An amino acid sequence that has the same function as the amino acid sequence shown in (I) or (II) obtained by modifying, substituting, deleting or adding one or more amino acids.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention includes a monoclonal antibody, a chimeric antibody, a humanized antibody or a murine antibody, and the antigen-binding fragment includes Fab, Fab', F(ab')2, scFv or scFv Fc fragments.

[0009] A third aspect of the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a neoantigen peptide isolated according to the first aspect of the invention or an antibody or antigen-binding fragment thereof according to the second aspect of the invention.

[0010] In a fourth aspect, the invention provides a carrier molecule comprising the nucleic acid molecule described in the third aspect of the invention.

[0011] In a fifth aspect, the invention provides a host cell comprising the nucleic acid molecule described in the third aspect of the invention or the carrier molecule described in the fourth aspect of the invention.

[0012] A sixth aspect of the present invention provides a method for preparing the isolated neoantigen peptide according to the first aspect of the present invention or the antibody or antigen-binding fragment thereof according to the second aspect of the present invention, wherein the isolated neoantigen peptide or the antibody or antigen-binding fragment thereof is prepared by artificial synthesis or genetic engineering.

[0013] A seventh aspect of the invention provides a detection product for the neuronal intranuclear inclusion body disease pathogenic protein PolyGN2C-iso2, comprising the antibody or antigen-binding fragment thereof described in the first aspect of the invention.

[0014] In some embodiments, the detection product according to the present invention includes a reagent kit, test strip, or protein chip.

[0015] An eighth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention in the preparation of a detection product for the neuronal intranuclear inclusion body disease pathogenic protein PolyGN2C-iso2 or a therapeutic agent for neuronal intranuclear inclusion body disease.

[0016] The antibody of this invention can specifically recognize the pathogenic protein PolyGN2C-iso2 in neuronal intranuclear inclusion body disease, and exhibits extremely low cross-reactivity with uN2CpolyG, resulting in very high accuracy. Therefore, it facilitates the diagnosis of neuronal intranuclear inclusion body disease with high specificity and high sensitivity. Furthermore, the monoclonal antibody of this invention is derived from stable cell clones, allowing for long-term culture and amplification, ensuring product quality stability, and enabling its use in the treatment of neuronal intranuclear inclusion body disease. Attached Figure Description

[0017] Figure 1 The results of the immunogenicity test for antigen Pep3 are shown.

[0018] Figure 2 The purification results of the monoclonal antibody against antigen Pep3 of the present invention are shown.

[0019] Figure 3 The affinity test results for the antibody Anti-A-N2C-Pep3-KLH:3 of the present invention are shown.

[0020] Figure 4 A schematic diagram of antigenic epitope screening for the novel pathogenic protein PolyGN2C-iso2 is shown.

[0021] Figure 5 The immunogenicity test results for Pep1, Pep2, and Pep3 are shown. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Product testing In one aspect, the present invention provides a detection product for detecting (or quantifying) the neuronal intranuclear inclusion body disease pathogenic protein PolyGN2C-iso2 in a sample, comprising an antibody or an antigen-binding fragment thereof, and instructions on how to implement the detection method of the present invention for the neuronal intranuclear inclusion body disease pathogenic protein PolyGN2C-iso2, wherein the antibody or the antigen-binding fragment thereof is capable of specifically binding to the neuronal intranuclear inclusion body disease pathogenic protein PolyGN2C-iso2, and the antibody or the antigen-binding fragment thereof comprises the heavy chain CDR1-3 shown in SEQ ID NO. 1-3 and / or the light chain CDR1-3 shown in SEQ ID NO. 4-6.

[0026] The present invention also provides a detection product for neuronal intranuclear inclusion body disease, which comprises the antibody or antigen-binding fragment thereof described in the present invention.

[0027] In this invention, the sample is not particularly limited, and examples include, but are not limited to, blood, skin tissue, and nerve tissue biopsy samples. Detection can be performed using methods known in the art, such as, but not limited to, immunohistochemistry (IHC), immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), Western blotting, and flow cytometry.

[0028] In a preferred embodiment, the detection product includes a reagent kit, test strip, or protein chip.

[0029] In this document, the term "kit" refers to a combination of reagents and other materials. A kit is intended to contain reagents such as buffers, protein stabilizing agents, signal generation systems (e.g., fluorescence signal generation systems), antibodies or antigen-binding fragments thereof, control proteins, and test containers (e.g., microtiter plates, etc.). The term "kit" is not limited to a specific combination of reagents and / or other materials; for example, a kit may also include instructions for using the reagents. Kits can be packaged in any suitable manner, typically having components in a single container or (if necessary) in multiple containers, along with instructions for performing the assay. Kits can be prepared using a variety of methods known in the art.

[0030] In some embodiments, the kit may further include at least one of a washing solution, a substrate solution, a diluent, and a calibration solution. The washing solution is not particularly limited in composition, but examples include, but are not limited to, buffer solutions, surfactants, and preservatives. The substrate solution may use known substrates, including but not limited to chromogenic substrates, fluorescent substrates, and luminescent substrates. The diluent is not particularly limited in composition, but examples include, but are not limited to, buffer solutions and surfactants. The calibration solution is not particularly limited in composition, but examples include, but are not limited to, BSA solution, trehalose solution, and animal serum.

[0031] In this invention, examples of reagent kits include, but are not limited to, ELISA detection kits, plate-based chemiluminescence detection kits, fully automated chemiluminescence detection kits, radioimmunoassay kits, fluorescence immunoassay kits, chemiluminescence immunoassay kits, immunoblotting kits, flow cytometry detection kits, colloidal gold detection kits, bioluminescent immunoassay kits, immunochromatographic detection kits, latex-based kits, and kits based on direct or indirect competitive methods.

[0032] The present invention further provides the application of the antibody or its antigen-binding fragment described herein in the preparation of a detection product for the neuronal intranuclear inclusion body disease pathogenic protein PolyGN2C-iso2.

[0033] This invention also provides the application of the antibody or its antigen-binding fragment described herein in the preparation of a detection product for neuronal intranuclear inclusion body disease.

[0034] Isolated neoantigen peptides Recent studies have shown that GGC amplification of NOTCH2NLC in the context of transcript 2 can induce cytotoxicity or trigger some pathological phenotypes in animal models. However, these studies have not identified the specific pathogenic protein produced by transcript 2 in NIID patients, nor have they demonstrated that this protein can independently induce the core pathological features of NIID, nor have they proposed specific technical solutions for using it as a diagnostic and therapeutic target. Single-protein pathogenic models fail to fully explain the complex clinicopathological features of NIID, and the role of another major transcript variant 2 of the NOTCH2NLC gene in the disease remains unclear. Therefore, a more comprehensive understanding of the pathogenesis of NIID is urgently needed in this field to develop truly effective diagnostic and therapeutic tools.

[0035] In one aspect of the present invention, an isolated neoantigen peptide is provided, which is the neuronal intranuclear inclusion body pathogenic protein PolyGN2C-iso2, said neoantigen peptide having the amino acid sequence shown in SEQ ID NO.12.

[0036] The design process of the novel antigenic peptide of this invention is as follows: Three epitopes (Pep1, M38D5, and M11F6) of the PolyGN2C-iso2 protein were selected as screening targets. The amino acid sequences of Pep1, M38D5, and M11F6 are shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively. Antibodies were used to test the above three antigenic proteins on the skin tissue of NIID patients, and the results are as follows: Figure 5 As shown, M38D5 Ab and M11F6 Ab positive signals can be detected in p62 positive intranuclear inclusion bodies, but Pep1 Ab positive signal cannot be detected.

[0037] Antibody or its antigen-binding fragment In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of binding with high affinity and targeting to the neuronal intranuclear inclusion body disease pathogenic protein PolyGN2C-iso2, without being bound by any theory. The antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain CDR1-3 shown in SEQ ID NO. 1-3 and / or the light chain CDR1-3 shown in SEQ ID NO. 4-6, wherein the amino acid sequences of the heavy chain CDR1-3 are RYGMS (SEQ ID NO. 1), SFSSGVTTYYPDSVKG (SEQ ID NO. 2) and GRRYYAMDY (SEQ ID NO. 3), respectively; and the amino acid sequences of the light chain CDR1-3 are KSSQSLLDSDGRTYLS (SEQ ID NO. 4), LVSTLDS (SEQ ID NO. 5) and WQGTHFPQT (SEQ ID NO. 6), respectively.

[0038] In one specific embodiment, the antibody or its antigen-binding fragment has any one of the amino acid sequences shown in (I)-(III): (I) The heavy chain variable region sequence shown in SEQ ID NO.7 and / or the light chain variable region sequence shown in SEQ ID NO.8; (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function; (III) An amino acid sequence that has the same function as the amino acid sequence shown in (I) or (II) obtained by modifying, substituting, deleting or adding one or more amino acids.

[0039] In this article, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies typically contain variable and constant regions in each of their heavy and light chains. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors. Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antibody moiety that binds to the antigen.

[0040] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of this invention consists of a "framework" region interspersed with three "complementarity-determining regions (CDRs)". The framework regions are used to modulate the CDRs for specific binding to antigenic epitopes. The CDRs contain the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0041] In this document, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequences of this invention. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0042] In this document, the modified antibody sequences also fall within the scope of protection of this invention. The term "modification" refers to any chemical modification of an amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids with different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to a change in the amino acid sequence resulting in an increase of one or more amino acids compared to a naturally occurring molecule. It should be noted that in the modified antibodies provided by this invention, the modification preferably occurs in regions other than the variable region, such as the frame region or constant region of the antibody, and the modified antibody still retains the desired functional properties of the antibody or its antigen-binding fragment of this invention, or has improved antigen-binding properties.

[0043] In this invention, conserved amino acid substitutions are preferred, and these conserved antibody variants are preferably generated by amino acid substitutions according to Table 1: Table 1 In this invention, the antibody includes monoclonal antibody, chimeric antibody, humanized antibody, or murine antibody.

[0044] The term "monoclonal antibody," as used in this article, sometimes also called "monoclonal antibody" or "Ab," refers to an immunoglobulin derived from a pure cell line, possessing the same structure and chemical properties, and specific for a single antigenic determinant. Monoclonal antibodies differ from conventional polyclonal antibody preparations (which typically contain different antibodies targeting different determinants); each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained through hybridoma or recombinant engineered cell culture, free from contamination by other immunoglobulins. This characteristic contrasts with polyclonal antibody products, which generally consist of antibodies targeting different antigenic determinants. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a homogeneous group of antibodies, but this should not be interpreted as requiring any special methods to produce the antibody.

[0045] In this article, the term "chimeric antibody" generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to, or belongs to, a corresponding amino acid sequence from an antibody of a specific species, while the remaining segments of that chain are homologous to a corresponding sequence from another species. For example, the variable regions of both the light and heavy chains may originate from the variable region of an antibody from one animal species (e.g., mouse, rat, etc.), while the constant region is homologous to an antibody sequence from another species (e.g., human). For instance, to obtain a chimeric antibody, the variable region can be generated using non-human B cells or hybridoma cells, with the constant region combined with it derived from human cells. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region it is combined with. Furthermore, because the constant region of a chimeric antibody can be derived from humans, the likelihood of the chimeric antibody eliciting an immune response upon injection is lower than with antibodies using a non-human-derived constant region.

[0046] In this article, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences derived from non-human immunoglobulins, thereby reducing the immunogenicity of the xenobiotic antibody when introduced into humans, while maintaining the antibody's complete antigen-binding affinity and specificity.

[0047] In this document, the term "mouse antibody" generally refers to an antibody whose variable region framework and CDR region are derived from mouse germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences. In this invention, mouse antibodies may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced through in vitro random or point mutations or through in vivo somatic mutations.

[0048] In this article, the term "antigen-binding fragment" generally refers to one or more fragments in an antibody that perform the function of specifically binding antigens. The antigen-binding function of an antibody can be achieved through the full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved through a heavy chain containing fragments including Fv, scFv, dsFv, Fab, Fab', or F(ab')2, or a light chain containing fragments including Fv, scFv, dsFv, Fab, Fab', or F(ab')2.

[0049] In this invention, the terms “targeting,” “binding,” “immunobinding,” “specific binding,” and “targeting” are used interchangeably and generally refer to a non-covalent interaction occurring between an immunoglobulin molecule and an antigen specific to said immunoglobulin. The strength or affinity of an immunobinding interaction can be expressed as a dissociation constant (Kd), where a smaller Kd represents a higher affinity. “Affinity” refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, when used herein, “binding affinity” refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally expressed as a binding dissociation equilibrium constant. Affinity can be measured by methods commonly known in the art, including those known in the prior art and those described herein.

[0050] Unless otherwise stated, the antigens, antibodies, or antigen-binding fragments thereof described herein are isolated antigens, antibodies, or antigen-binding fragments thereof. The term "isolated" as used herein means an antigen, antibody, or antigen-binding fragment thereof that has been extracted from its natural environment. "Isolated" antigens, antibodies, or fragments thereof therefore include antigens, antibodies, or fragments thereof purified by standard purification methods. This term also includes antigens, antibodies, or fragments thereof prepared through recombinant expression in host cells, as well as chemically synthesized antigens, antibodies, or fragments thereof.

[0051] Nucleic acid molecules In one aspect of the present invention, a nucleic acid molecule is provided comprising a nucleotide sequence encoding a novel antigenic peptide or antibody or an antigen-binding fragment thereof isolated according to the present invention.

[0052] As used in this invention, the term "nucleic acid" is intended to include polymeric forms of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or their analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0053] Once the coding sequence of the neoantigen peptide or antibody described in this invention is isolated, recombinant technology can be used to obtain the neoantigen peptide or antibody in large quantities. An exemplary method is to clone its coding gene into a vector, transform it into cells, and then isolate it from the proliferated host cells using conventional methods.

[0054] carrier molecules In one aspect, the present invention provides a carrier molecule comprising the nucleic acid molecule described herein.

[0055] The vector of this invention refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The vector of this invention is not limited and can be an expression vector, viral vector, etc. In some embodiments, the vector contains a target gene encoding the antibody of this invention, a promoter, a terminator, or optionally a marker gene. The vector can be a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, etc.

[0056] host cells In one aspect, the present invention provides a host cell comprising the nucleic acid molecule or the carrier molecule described in the present invention.

[0057] The host cell of this invention refers to any cell type suitable for transformation, transfection, transduction, etc., using a nucleic acid construct or expression vector containing the nucleic acid molecules of this invention. The host cell includes any offspring of the parent cell that differs from the parent cell due to mutations occurring during replication.

[0058] Preparation method In one aspect, this invention provides a method for preparing the novel antigenic peptide or antibody or its antigen-binding fragment described herein. The preparation method is not particularly limited, and includes preparation by artificial synthesis or genetic engineering.

[0059] In some embodiments, the neoantigen peptides or antibodies of the present invention are obtained through artificial synthesis. Methods for artificially synthesizing neoantigen peptides or antibodies are known in the art, for example, the neoantigen peptides or antibodies of the present invention are obtained through direct amino acid synthesis.

[0060] In some embodiments, the neoantigen peptides or antibodies of the present invention are obtained through genetic engineering expression. Genetic engineering expression systems for use include, but are not limited to, prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include enzyme expression systems, insect cell expression systems, and mammalian cell expression systems.

[0061] In a preferred embodiment, the novel antigenic peptide or antibody of the present invention can be prepared by the following steps: (1) Construct a recombinant vector expressing the antibody heavy chain of the amino acid sequence shown in SEQ ID NO.7 and / or the antibody light chain of the amino acid sequence shown in SEQ ID NO.8, or a recombinant vector expressing the neoantigen peptide of the amino acid sequence shown in SEQ ID NO.12; (2) The vector is transformed into host cells and cultured under conditions suitable for antibody or neoantigen peptide expression; (3) Collect antibodies or neoantigen peptides and purify them.

[0062] In another preferred embodiment, the antibody of the present invention is prepared by immunization with an antigen having the amino acid sequence shown in SEQ ID No. 12.

[0063] Example This embodiment illustrates the preparation method and detection process of a monoclonal antibody targeting the neuronal intranuclear inclusion body pathogenic protein PolyGN2C-iso2.

[0064] 1. Antigen design Three epitopes (Pep1, M38D5, and M11F6) of the PolyGN2C-iso2 protein (SEQ ID NO. 9) were selected as screening targets. The amino acid sequences of Pep1, M38D5 (Pep2), and M11F6 (Pep3) are shown in SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12, respectively. Antibodies were used to test these three antigen proteins on skin tissue from NIID patients, and the results are as follows: Figure 5 As shown, M38D5 Ab and M11F6 Ab positive signals can be detected in p62 positive intranuclear inclusion bodies, but Pep1 Ab positive signal cannot be detected.

[0065] Using the M11F6 protein sequence of PolyGN2C-iso2, the pathogenic protein of neuronal intranuclear inclusion body disease, as the antigen sequence, KLH was conjugated and quantified at 3 mg / ml, and named N2C-pep3-KLH.

[0066] 2. Immunity 2.1 Animal Immunization (1) Four SPF-grade BALB / c female mice were subcutaneously immunized with “N2C-pep3-KLH” at a dose of 60 μg protein per mouse. The mice were numbered 1, 2, 3, and 4.

[0067] (2) Two weeks later, the first subcutaneous booster immunization was given, with an immunization dose of 30 μg protein per animal.

[0068] (3) Two weeks later, a second subcutaneous booster immunization was administered, with an immunization dose of 30 μg protein per animal.

[0069] (4) Two weeks later, a third subcutaneous booster immunization was administered, with an immunization dose of 30 μg protein per animal.

[0070] (5) One week later, blood was drawn from the eye socket to measure the serum titer.

[0071] (6) Four days later, #4 mice were intraperitoneally induced with 50 μg of immunogen.

[0072] 2.2 Immunotiter determination The serum was coated overnight at 4°C with N2C-pep3-BSA at 2 μg / ml; blocked with 2% milk at 37°C for 2 h; serum was serially diluted 2-fold starting from 200-fold, with PBS as the blank control and negative serum diluted 200-fold as the negative control. The immunotiter test results are shown in Table 2. Figure 1 As shown in the figure. Impact #4 mice were selected for cell fusion experiments.

[0073] Table 2 Results of Immunotiter Test 2.3 Cell Fusion Experiment Mouse spleen cells were fused with SP20 cells using the PEG method. After fusion, the cells were cultured in a semi-solid medium (containing HAT) for selection.

[0074] 2.3.1 Experimental Equipment Sterilized surgical instruments: three pairs of scissors, three pairs of forceps, a cell strainer, a syringe insert, and a petri dish. A humidified chamber, two 500 ml beakers, two 50 ml centrifuge tubes, and three 15 ml centrifuge tubes.

[0075] 2.3.2 Experimental Reagents IMDM medium; IMDM complete medium (containing 15% serum); 2.3% methylcellulose (SIGMA, M0262-100G); newborn calf serum: 10 ml; PEG1450 (Sigma, P7181); HAT (Sigma, H0262-10VL); HT (Sigma, H0137-10VL).

[0076] 2.3.3 Fusion Experiment Procedure 1) Gently pipette healthy sp2 / 0 cells off the culture flask wall and transfer them into a 50 ml centrifuge tube.

[0077] 2) Blood was collected from the eyes of the mice, and they were then euthanized by pulling their necks. The mice were then soaked in 75% alcohol for 5 minutes.

[0078] 3) Pour a small amount of serum-free IMDM into a petri dish, and place the cell strainer and syringe core into the dish. Remove the mouse spleen with scissors and forceps and place it on the cell strainer. Gently crush the spleen thoroughly with the syringe core, and aspirate the crushed cells into a centrifuge tube containing sp2 / 0. Centrifuge at 1500 rad / min for 5 min.

[0079] 4) Remove the mouse thymus with scissors and tweezers and crush it. Transfer the crushed thymus cells to a 15 ml centrifuge tube, add 2 ml of HAT and 1 ml of HT, and place in an incubator for later use.

[0080] 5) After centrifuging, discard the supernatant, carefully and gently reconstitute the cells with serum-free IMDM, and centrifuge (1500 rad / min, 5 min).

[0081] 6) Discard as much of the cell supernatant as possible after centrifugation. Gently tap the bottom of the centrifuge tube to fully resuspend the cells. Place the centrifuge tube in 37°C warm water. Slowly add 1 ml of PEG over approximately 1 minute. After adding the PEG, let the tube stand in the warm water for 1 minute. Then, slowly add 2 ml of serum-free IMDM over 2 minutes, followed by 8 ml of serum-free IMDM over another 2 minutes. Centrifuge at 1000 rad / min for 5 minutes.

[0082] 7) Discard the supernatant, add 10 ml of serum, carefully mix the cells by blowing, and pour into the prepared thymocytes. Add sterile semi-solid culture medium to a final volume of 50 ml and mix thoroughly. Then pour evenly into 30 cell culture dishes. Place the cell culture dishes in a humidified chamber and then incubate in an incubator.

[0083] 2.4 Selecting Clones Select 10 plates × 93 cell clones and culture them in 96-well cell culture plates (pre-coated with thymocytes, 100 μl / well).

[0084] 2.5 Single-clonal cell screening Discard all supernatant from the monoclonal cells in the 96-well culture plate, add 200 μl / well of 20% newborn calf IMDM medium (containing HT), and perform the first screening.

[0085] 2.6 Monoclonal cell screening Using "N2C-pep3-BSA" plates, the selected clones were screened a second time using the ELISA method, resulting in 24 positive hybridoma cell lines.

[0086] 2.6.1 Experimental Reagents Coating buffer: Sodium carbonate-sodium bicarbonate buffer, pH=9.6; PBS buffer: pH=7.4; Blocking buffer: PBS with 2% skim milk powder; Washing buffer: PBS-T (0.05% Tween, PBS); Chromogenic solution: 1% solution A + 10% solution B (Solution A: DMSO with 1% TMB; Solution B: Citrate buffer with 0.1% CH4N2O·H2O2); Stop solution: 0.5 M sulfuric acid; Secondary antibody: Goat anti-mouse IgG / HRP.

[0087] 2.6.2 Experimental Procedure 1) Dilute “N2C-pep3-BSA” with coating buffer to a final concentration of 2 μg / ml, 100 μl / well, incubate overnight at 4℃, and then wash 3 times with washing buffer.

[0088] 2) Block with blocking solution (2% skim milk powder), 200 μl / well, incubate at 37℃ for 2 h, then wash 3 times with washing solution.

[0089] 3) Add 100 μl of primary antibody (cell culture supernatant), negative control (SP2 / 0 culture supernatant), blank control (PBS), and positive control (positive serum diluted 1000 times with PBS) to each well. Incubate at 37°C for 1 h, and then wash 3 times with washing buffer.

[0090] 4) Add PBS to dilute the secondary antibody 20,000 times, 100 μl / well, incubate at 37℃ for 1 h, and wash 3 times with washing buffer after taking it out.

[0091] 5) Add 100 μl of colorimetric solution per well, and the color development time is about 10 min.

[0092] 6) Add 50 μl of stop solution to each well to terminate the process.

[0093] 7) Measure the absorbance at dual wavelengths (450, 630), record and save the data, as shown in Table 3.

[0094] Table 3. Data from the second screening experiment of hybridoma cells 2.7 Monoclonal cell 3-stage screening The 24 positive cell lines were coated again with "N2C-pep3-BSA" and screened a third time using ELISA, resulting in 5 positive hybridoma cell lines. The experimental data are shown in Table 4.

[0095] Table 4. Data from the third screening experiment of hybridoma cell lines 2.8 Identification of Monoclonal Cell Subclasses 2.8.1 Experimental Reagents Subclass coating antibodies: (Goat Anti-Mouse Ig, Human ads-UNLB); Blocking buffer: PBS with 2% skim milk powder; Wash buffer: PBS-T (0.05% Tween, PBS); Subclass secondary antibodies: (Goat Anti-Mouse IgM, IgG1, IgG2a, IgG2b, etc.); Chromogenic solution: 1% solution A + 10% solution B (Solution A: DMSO with 1% TMB; Solution B: Citrate buffer with 0.1% CH4N2O·H2O2); Stop solution: 0.5 M sulfuric acid.

[0096] 2.8.2 Experimental Procedure 1) Dilute the “subclass coated antibody” with coating buffer to a final concentration of 2 μg / ml, 100 μl / well, incubate overnight at 4°C, and then wash 3 times with washing buffer.

[0097] 2) Block with blocking solution, 200 μl / well, incubate at 37℃ for 2 h, then wash 3 times with washing solution.

[0098] 3) Add 100 μl of primary antibody (cell culture supernatant) and negative control (SP2 / 0 culture supernatant) to each well, incubate at 37℃ for 1 h, and then wash 3 times with washing buffer.

[0099] 4) Dilute each subtype of secondary antibody with PBS, 100 μl / well, add to the appropriate well, incubate at 37°C for 1 h, and wash 3 times with washing buffer after removal.

[0100] 5) Add 100 μl of colorimetric solution per well, and the color development time is about 10 min.

[0101] 6) Add 50 μl of stop solution to each well to terminate the process.

[0102] 7) Measure the absorbance at dual wavelengths (450, 630), record and save the data. The results are shown in Table 5.

[0103] By subclassing the five positive cell lines selected, four IgG positive hybridoma cell lines were finally obtained, and the results are shown in Table 6.

[0104] Table 5. Experimental data for the identification of monoclonal cell subclasses Table 6. Results of Monoclonal Cell Subclass Identification 3. Antibody purification and detection 3.1 Purification Antibodies produced by four IgG-positive hybridoma cell lines were purified, and the data are shown in Table 7. Figure 2 As shown.

[0105] Table 7 Antibody Purification Data 3.2 ELISA detection The affinity constants of the antibodies were detected by ELISA, and the results are shown in Table 8. Figure 3 As shown.

[0106] Table 8. Antibody ELISA Detection Results 3.3 Sequencing Molecular sequencing was performed on the Anti-N2C gene-Pep3-KLH:3, and the sequence is shown in Table 9.

[0107] Table 9. Amino acid sequence of Anti-N2C gene-Pep3-KLH:3 Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or its antigen-binding fragment, characterized in that, It can specifically bind to the neuronal intranuclear inclusion body disease pathogenic protein PolyGN2C-iso2, which has the amino acid sequence shown in SEQ ID NO.

12. The antibody or its antigen-binding fragment includes the heavy chain CDR1-3 shown in SEQ ID NO.1-3 and the light chain CDR1-3 shown in SEQ ID NO.4-6.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment has an amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function, wherein the sequence shown in (I) has the heavy chain variable region sequence shown in SEQ ID NO.7 and the light chain variable region sequence shown in SEQ ID NO.

8.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The antibody or its antigen-binding fragment has the heavy chain variable region sequence shown in SEQ ID NO.7 and the light chain variable region sequence shown in SEQ ID NO.

8.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody includes a monoclonal antibody, and the antigen-binding fragment includes Fab, Fab', F(ab')2, scFv, or scFv Fc fragments.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The monoclonal antibodies include chimeric antibodies, humanized antibodies, or murine antibodies.

6. An isolated neoantigen peptide, characterized in that, It is the neuronal intranuclear inclusion body disease pathogenic protein PolyGN2C-iso2, and the neoantigen peptide has the amino acid sequence shown in SEQ ID NO.

12.

7. A nucleic acid molecule, characterized in that, It comprises a nucleotide sequence encoding the isolated neoantigen peptide of claim 6 or the antibody or antigen-binding fragment thereof of any one of claims 1-5.

8. A carrier molecule, characterized in that, It comprises the nucleic acid molecule as described in claim 7.

9. A host cell, characterized in that, It comprises the nucleic acid molecule of claim 7 or the carrier molecule of claim 8.

10. A method for preparing the isolated neoantigen peptide according to claim 6 or the antibody or antigen-binding fragment thereof according to any one of claims 1-5, characterized in that, The isolated neoantigen peptide or the antibody or its antigen-binding fragment are prepared by artificial synthesis or genetic engineering.

11. A detection product for the pathogenic protein PolyGN2C-iso2 in neuronal intranuclear inclusion body diseases, characterized in that, It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-5.

12. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-5 in the preparation of a detection product for the neuronal intranuclear inclusion body disease pathogenic protein PolyGN2C-iso2 or a medicament for treating neuronal intranuclear inclusion body disease.

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