MDA5 humanized monoclonal antibody and preparation method and application thereof

Abstract

The present application relates to a kind of MDA5 humanization monoclonal antibody and its preparation method and application.The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the antibody respectively include the sequences shown in SEQ ID NO:6~SEQ ID NO:8, the amino acid sequences of CDR1, CDR2 and CDR3 of light chain respectively include the sequence shown in SEQ ID NO:9, DNN and the sequence shown in SEQ ID NO:10.The present application successfully screens a human monoclonal antibody, which has high activity, good stability and precise specific binding capacity to MDA5, and is expected to become a new tool in the diagnosis, monitoring and treatment of dermatomyositis and its related complications, and bring new opportunities for the attack of these medical problems.

Description

Technical Field

[0001] This invention belongs to the field of bioimmunotechnology, and relates to an MDA5 humanized monoclonal antibody, its preparation method, and its application. Background Technology

[0002] Dermatomyositis is a rare inflammatory myopathy characterized by muscle weakness and skin lesions. Due to its complex etiology and diverse presentations, the diagnosis of dermatomyositis has always been a challenge in clinical medicine. Early diagnosis and accurate assessment are crucial for disease control and treatment outcomes. Traditional diagnostic methods for dermatomyositis mainly rely on clinical manifestations, muscle enzyme levels, and muscle biopsies. These methods suffer from drawbacks such as high invasiveness, complex procedures, and insufficient sensitivity and specificity. In recent years, with the development of molecular biology and genomics, researchers have discovered many biomarkers associated with dermatomyositis, including myositis-specific autoantibodies (MSAs) and interferon-related biomarkers. These biomarkers help to understand the pathological mechanisms of dermatomyositis and provide new insights for diagnosis and treatment.

[0003] Myositis-specific autoantibodies mainly include anti-Mi-2 antibodies, anti-transcriptional mediator 1-γ antibodies, MDA5 antibodies, and anti-matrix protein 2 antibodies. Melanoma differentiation-related genes 5 (MDA5) is an intracellular sensor of viral RNA (including coronaviruses) that can trigger an innate immune response. MDA5 antibodies can serve as biomarkers for dermatomyositis (DM) patients. It has been confirmed that this antibody is closely related to the development of interstitial lung disease (ILD) in DM patients. The presence of MDA5 antibodies in human serum is an important biomarker for diagnosing and predicting the prognosis of patients with idiopathic inflammatory myopathy. It can also serve as a biomarker for the clinical diagnosis of clinically amyopathic dermatomyositis (CADM). The presence of MDA5 antibodies is also associated with poor overall survival prognosis in DM-ILD patients.

[0004] Currently, the main detection methods for MDA5 antibodies include enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunofluorescence assay. These methods provide varying degrees of support for the detection of MDA5 antibodies, but they still have shortcomings such as insufficient sensitivity and specificity, operational complexity and time consumption, and limited dynamic monitoring capabilities.

[0005] In summary, there is currently a lack of effective detection methods for rapidly and accurately diagnosing diabetes mellitus and quantitatively detecting MDA5 antibody levels in patients' serum. Summary of the Invention

[0006] In view of the shortcomings of existing technologies and practical needs, this invention provides a humanized monoclonal antibody for MDA5, its preparation method and application, in order to be used for the identification of MDA5 antigen and to assist in the diagnosis and detection of MDA5 antibody-positive dermatomyositis or its complications.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a humanized monoclonal antibody for MDA5, wherein the amino acid sequence of CDR1 of the heavy chain of the antibody includes the sequence shown in SEQ ID NO:6, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID NO:7, the amino acid sequence of CDR3 includes the sequence shown in SEQ ID NO:8, the amino acid sequence of CDR1 of the light chain includes the sequence shown in SEQ ID NO:9, the amino acid sequence of CDR2 is DNN, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID NO:10.

[0009] In this invention, a phage library was constructed based on human samples and humanized antibodies against dermatomyositis-specific antigen (MDA5) were obtained through screening. These antibodies have high activity, good stability, and strong specificity. They can be used as a reference standard for qualitative detection of MDA5 positivity and can also quantitatively detect the level of anti-MDA5 autoantibodies in patients with amyopathy dermatomyositis, providing new tools and ideas for the diagnosis of dermatomyositis and its complications.

[0010] Preferably, the amino acid sequence of the heavy chain variable region of the antibody includes the sequence shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO:5.

[0011] Preferably, the heavy chain amino acid sequence of the antibody includes the sequence shown in SEQ ID NO:2, and the light chain amino acid sequence includes the sequence shown in SEQ ID NO:3.

[0012] SEQ ID NO:2:

[0013] MKHLWFFLLLVAAPRWVLSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSYGINWVRQAPGKGLEWVSGISASGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCANLRYDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0014] SEQ ID NO:3:

[0015] MVLQTQVFISLLLWISGAYGQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKFGTSATLAITGLQTGDEADYYCASWDNSLTAGVFGGGTQLTALGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS。

[0016] SEQ ID NO:4:

[0017] EVQLVESGGGLVQPGGSLRLSCAASGFTFRSYGINWVRQAPGKGLEWVSGISASGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCANLRYDFWGQGTMVTVSS。

[0018] SEQ ID NO:5:

[0019] QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKFGTSATLAITGLQTGDEADYYCASWDNSLTAGVFGGGTQLTAL.

[0020] SEQ ID NO:6:GFTFRSYG.

[0021] SEQ ID NO:7:ISASGGST.

[0022] SEQ ID NO:8: ANLRYDF.

[0023] SEQ ID NO:9: SSNIGNNY.

[0024] CDR2:DNN for light chains.

[0025] SEQ ID NO:10: ASWDNSLTAGV.

[0026] It is understood that, based on the antibodies screened in this invention, antibodies with similar functions obtained by using genetic modification methods in the art to perform amino acid substitution, deletion, or addition should all be within the scope of protection of this invention. The number of amino acids substituted, deleted, or added can be any value, such as 1, 5, 10, 15, or more, such that the sequence identity between the changed amino acid sequence and its corresponding original sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In the art, when amino acids with similar or comparable properties are conservatively substituted (conservative mutations), the function of the protein is usually not changed. For example, amino acids with similar properties are substituted in the CDR region and / or FR region. The amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. Therefore, antibodies obtained by conservatively substituting amino acids with similar or comparable properties are also within the scope of protection of this invention.

[0027] In a second aspect, the present invention provides a nucleic acid molecule that encodes the MDA5 humanized monoclonal antibody described in the first aspect.

[0028] Thirdly, the present invention provides a recombinant expression vector containing the nucleic acid molecule described in the second aspect.

[0029] In this invention, due to the degeneracy of the genetic code, a large number of nucleic acid molecules that can be used to encode the antibodies of this invention can be obtained. Therefore, given the identification of specific amino acid sequences, those skilled in the art can easily prepare any number of different nucleic acids by modifying the sequences of one or more codons without altering the amino acid sequence encoding the protein. More preferred polynucleotides can be selected through codon optimization based on the preferences of the host cells used in the actual preparation process. The nucleic acid molecules can be obtained using conventional methods, such as PCR amplification or artificial synthesis.

[0030] Fourthly, the present invention provides an engineered cell containing the nucleic acid molecules described in the second aspect.

[0031] The engineered cell expression of the MDA5 humanized monoclonal antibody described in the first aspect of the present invention may contain a nucleic acid molecule encoding the antibody or a recombinant vector containing the nucleic acid molecule. The host cell may be a prokaryotic cell, a lower eukaryotic cell, or a higher eukaryotic cell. Prokaryotic cells may be bacterial cells, lower eukaryotic cells may be yeast cells, and higher eukaryotic cells may be mammalian cells.

[0032] Fifthly, the present invention provides a method for preparing the humanized monoclonal antibody MDA5 described in the first aspect, the method comprising:

[0033] The nucleic acid sequence encoding the humanized monoclonal antibody MDA5 described in the first aspect is transduced into a host cell and expressed, and then purified to obtain the humanized monoclonal antibody MDA5.

[0034] Transformation of nucleic acid expression vectors into host cells can be performed using conventional methods well-known to those skilled in the art. These include methods such as CaCl2 injection, electroporation, calcium phosphate co-precipitation, and conventional mechanical methods like microinjection, electroporation, and liposome packaging. The resulting transformants can be cultured using conventional methods well-known to those skilled in the art, and the culture medium can be a standard culture medium. Antibodies produced by the transformants can be separated and purified using physical and chemical methods, such as salting out, centrifugation, cell disruption, and chromatography.

[0035] In a sixth aspect, the present invention provides the use of the MDA5 humanized monoclonal antibody described in the first aspect in any one of the following (1)-(5):

[0036] (1) Prepare reagents or kits for detecting MDA5;

[0037] (2) Prepare reagents or kits for diagnosing dermatomyositis or its complications;

[0038] (3) Detection of MDA5 for non-diagnostic and non-therapeutic purposes;

[0039] (4) Prepare quality control materials for MDA5 antibody detection;

[0040] (5) Prepare a composition for tracing.

[0041] Preferably, the dermatomyositis includes MDA5 antibody-positive dermatomyositis.

[0042] In a seventh aspect, the present invention provides a reagent or kit comprising the MDA5 humanized monoclonal antibody described in the first aspect.

[0043] Preferably, the reagent includes at least one of enzyme-linked immunosorbent assay (ELISA) reagent, immunoblotting reagent, or immunohistochemical assay reagent.

[0044] Eighthly, the present invention provides a method for detecting MDA5 for non-diagnostic and therapeutic purposes, the method comprising detecting a sample to be tested using the humanized monoclonal antibody against MDA5 described in the first aspect.

[0045] In a ninth aspect, the present invention provides an antibody conjugate comprising the MDA5 humanized monoclonal antibody described in the first aspect and a conjugating substance thereto.

[0046] Preferably, the coupling substance includes at least one of cytotoxin, radioactive isotope, luminescent material, chromogenic substance or enzyme.

[0047] Compared with the prior art, the present invention has at least the following beneficial effects:

[0048] This invention screens and obtains a humanized monoclonal antibody against MDA5. This antibody has high activity, good stability, and strong specificity for binding to MDA5. It can be used as a reference standard for qualitative detection of MDA5 positivity, and can also be used for quantitative detection of anti-MDA5 autoantibody levels in patients with inflammatory myopathy or its complications. It provides effective evidence for the diagnosis, disease monitoring, prognostic assessment, and disease mechanism research of patients with DM, CADM, DM combined with ILD, and CADM combined with ILD. Attached Figure Description

[0049] Figure 1 This is an agarose gel electrophoresis image of a single clone of a VL phage library.

[0050] Figure 2 This is an agarose gel electrophoresis image of a single clone of the KH phage library.

[0051] Figure 3 This is an agarose gel electrophoresis image of a single clone of the λH phage library.

[0052] Figure 4This is an SDS-PAGE electrophoresis image of the recombinant protein MDA5.

[0053] Figure 5 The image shows the ELISA results for different concentrations of MDA5 antibody diluted.

[0054] Figure 6 This is a Western blot result of the MDA5 antibody. Detailed Implementation

[0055] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0056] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0057] Unless otherwise defined, scientific and technical terms and their abbreviations used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Some of the terms and abbreviations used in this invention are listed below.

[0058] The term "dermatomyositis," also known as "DM," is a non-suppurative inflammatory condition that primarily affects skeletal muscle, characterized by predominantly lymphocytic infiltration, and may or may not be accompanied by various skin lesions. The terms "dermatomyositis" and "DM" are used interchangeably.

[0059] The term "clinically amyopathic dermatomyositis" (CADM) refers to a type of dermatomyositis characterized by skin lesions alone or predominantly skin lesions. "Clinically amyopathic dermatomyositis" and "CADM" are used interchangeably.

[0060] The term "interstitial lung disease" (ILD) refers to inflammatory diseases of the lung interstitium caused by various factors. The lesions primarily affect the lung interstitium, but can also involve alveolar epithelial cells and pulmonary blood vessels. "Interstitial lung disease" and "ILD" are used interchangeably.

[0061] The term "acute interstitial pneumonia" (AIP) is a rapidly developing fulminant lung injury, an acute traumatic lesion of the lungs. "Acute interstitial pneumonia" and "AIP" are used interchangeably.

[0062] The terms "MDA5," "MDA5 antigen," and "MDA5 protein" are used interchangeably. MDA5 has two N-terminal pattern recognition receptors (caspase activation and recruitment domains, CARDs) and one RNA helicase domain. The CARD domain is responsible for signal transduction via Toll-like receptors, while the helicase domain is responsible for recognizing viral RNA. Virus-infected cells, especially fibroblasts, dendritic cells, and macrophages, highly express MDA5, which can recognize virus-derived nucleic acid molecules in the cytoplasm, initiate the type I interferon pathway, and lead to the production of a series of inflammatory mediators.

[0063] The term "conserved mutation" refers to a mutation (e.g., amino acid substitution, insertion, and / or deletion) that maintains the normal function of a protein.

[0064] The terms “sequence identity” and “identity percentage” refer to the percentage of identical (i.e., the same) nucleotides or amino acids between two or more polynucleotides or polypeptides.

[0065] The term “antibody” is used in the broadest sense herein to refer to a protein that contains an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, intact antibodies, and antibody fragments.

[0066] The terms "complementarity-determining region" or "CDR region" or "CDR" refer to regions within the antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariant loops") and / or contain antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes. Heavy and light chain CDRs are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. Within a given amino acid sequence of a light or heavy chain variable region, the precise amino acid sequence boundaries of each CDR can be determined using any or a combination of many known antibody CDR assignment systems.

[0067] The main reagents involved in the specific embodiments of this invention are shown in Table 1.

[0068] Table 1

[0069]

[0070] Example 1

[0071] This embodiment describes the construction of a human phage display library.

[0072] RNA from MDA5 antibody-positive patients was reverse transcribed into cDNA using the HiScript® III first-strand cDNA synthesis kit, and the VH and VL fragments of the DNA were amplified. The VK and Vλ gene fragments obtained in vitro were cloned into the pATA-scFv-2 vector to form VK and Vλ libraries, respectively. Plasmid vectors for the VK and Vλ libraries were extracted using a plasmid extraction kit, and the in vitro amplified VH gene fragment was inserted into these vectors to form KH and λH libraries, respectively.

[0073] 1. Library Construction

[0074] 1.1 Assemble the heavy chain variable region (VH) and the light chain variable region (VL).

[0075] The PCR reaction conditions and steps are shown in Table 2.

[0076] Table 2

[0077]

[0078] The three steps of denaturation, annealing, and extension (1) are repeated 30 times.

[0079] Primer sequences:

[0080] upstream primer (F) for the heavy chain variable region:

[0081] 5'L-VH 1 (SEQ ID NO: 13): acaggtgcccactcccaggtgcag.

[0082] 5'L-VH 3 (SEQ ID NO: 14): aaggtgtccagtgtgargtgcag.

[0083] 5'L-VH 4 / 6 (SEQ ID NO: 15): cccagatgggtcctgtcccaggtgcag.

[0084] 5'L-VH 5 / 7 (SEQ ID NO:16):caaggagtctgttccgaggtgcag.

[0085] upstream primer (F) of the light chain variable region:

[0086] 5'L VK 1 / 2 (SEQ ID NO:17): atgaggstcccygctcagctgctgg.

[0087] 5'L VK 3 (SEQ ID NO: 18): ctcttcctcctgctactctggctcccag.

[0088] 5'L VK 4 / 5 (SEQ ID NO:19): atttctctgttgctctggatctctg.

[0089] 5'L Vλ 1 (SEQ ID NO:20): ggtcctgggcccagtctgtgctg.

[0090] 5' L Vλ 2 (SEQ ID NO:21): ggtcctgggcccagtctgccctg.

[0091] 5'L Vλ 3 (SEQ ID NO:22): gctctgtgacctcctatgagctg.

[0092] 5'L Vλ 4 / 5 (SEQ ID NO:23): ggtctctctcscagcytgtgctg.

[0093] 5'L Vλ 6 (SEQ ID NO:24): gttcttgggccaattttatgctg.

[0094] 5'L Vλ 7 (SEQ ID NO:25): ggtccaattcycaggctgtggtg.

[0095] 5'L Vλ 8 / 9 / 10 (SEQ ID NO:26): gagtggattctcagactgtggtg.

[0096] Downstream primer (R) for the light chain variable region:

[0097] 3'CK (SEQ ID NO:27): tgctgtccttgctgtcctgct.

[0098] 3'Cλ (SEQ ID NO:28): caccagtgtggccttgttggcttg.

[0099] Downstream primer (R) for the heavy chain variable region:

[0100] 3'VH (SEQ ID NO:29):ACTCGAGACGGTGACCAGGGTGCC.

[0101] 1.2 Construction of a light chain variable region phage display library

[0102] 1.2.1 Preparing the pATA-scFv-2 vector for library cloning

[0103] 1.2.2 Digestion of vectors and PCR products

[0104] The reaction system for digesting the vector and PCR product is shown in Table 3.

[0105] Table 3

[0106]

[0107] 1.2.3 Connection

[0108] The connection reaction system is shown in Table 4. It was incubated overnight at 16°C and then inactivated by heating at 65°C for 10 min.

[0109] Table 4

[0110]

[0111] 1.2.4 Electrical Transfer

[0112] 1) Preparation of TG1 competent cells

[0113] 2) Preheat 1 mL of SOC medium (Sigma, S1797) to 37°C. Place the electroporation cuvettes (0.1 cm gap) and microcentrifuge tubes on ice (one cuvette and one microcentrifuge tube per conversion reaction).

[0114] 3) Remove the electrocompetent cells from the -80°C freezer and place them on ice until they are completely thawed. After thawing, gently mix the cells. Transfer 50 μL of cells to a frozen microcentrifuge tube placed on ice.

[0115] 4) Carefully add 3 μL of the DNA mixture to a frozen electroporation cuvette, allowing the cells to settle at the bottom.

[0116] 5) Electroporation was performed at 600 Ω, 10 μF, and 1.8 kV. Within 10 s of the pulse, 1 mL of preheated SOC medium was added to each test tube immediately. The tubes were then incubated at 37°C and shaken at 250 rpm for 1 h.

[0117] 6) Collect all electroporation media. Serially dilute 10 μL of culture into 90 μL of SOC medium and spread it onto LB / Amp / glucose solid medium. Incubate overnight at 37°C. Calculate the total number of transformants by counting the number of colonies, multiplying by the culture volume, and dividing by the inoculation volume.

[0118] 1.3 Construction of VL-VH phage display library

[0119] 1.3.1 Digestion of vectors and PCR products

[0120] The digestion reaction system is shown in Table 5.

[0121] Table 5

[0122]

[0123] 1.3.2 Connection

[0124] The connection reaction system is shown in Table 6. It was incubated overnight at 16°C and then inactivated by heating at 65°C for 10 min.

[0125] Table 6

[0126]

[0127] 1.3.3 Electrical Transfer

[0128] 1) Preparation of TG1 competent cells.

[0129] 2) Preheat 4 mL of SOC medium (Sigma, S1797) to 37°C. Place the electroporation cuvettes (0.2 cm gap) and microcentrifuge tubes on ice (one cuvette and one microcentrifuge tube per conversion reaction).

[0130] 3) Remove the electrocompetent cells from the -80°C freezer and place them on ice until they are completely thawed. After thawing, gently mix the cells.

[0131] 4) Add 6 μL of the DNA mixture to a frozen electroporation cuvette to allow the cells to settle at the bottom.

[0132] 5) Electroporation was performed at 600 Ω, 10 μF, and 2.5 kV. Within 10 seconds of the pulse, 2 mL of preheated SOC medium was immediately added to each tube. The tubes were then incubated at 37°C and shaken at 250 rpm for 1 h.

[0133] 6) Collect all electroporation media. Serially dilute 10 μL of culture into 90 μL of SOC medium and spread it onto LB / Amp / glucose solid medium. Incubate overnight at 37°C. Calculate the total number of transformants by counting the number of colonies, multiplying by the culture volume, and dividing by the inoculation volume.

[0134] 1.4 Library Evaluation

[0135] 1.4.1 Colony PCR: Using the constructed library as a template, PCR was performed. The PCR reaction conditions are shown in Table 7.

[0136] Table 7

[0137]

[0138] The denaturation, annealing, and extension (1) steps were repeated 30 times. Primer sequences: forward primer (F): AGCGGATAACAATTTCACACAGGA (SEQ ID NO:30); reverse primer (R): GCCCCTTATTAGCGTTTGCCATC (SEQ ID NO:31).

[0139] The results of agarose gel electrophoresis after PCR are as follows: Figures 1-3 As shown, Figure 1 This is an agarose gel electrophoresis image of monoclonal bacteria from the VL phage library. Lanes 1-16 are pATA-VK, and lanes 17-32 are pATA-Vλ. Figure 2 This is a PCR agarose gel electrophoresis image of a single clone of the KH phage library; lane M: DL2000, lanes 1-48 pATA-scFv-KH. Figure 3 This is an agarose gel electrophoresis image of monoclonal PCR of the λH phage library; lane M: DL2000, lanes 1-48 pATA-scFv-λH. Colony PCR results showed that the fragment sizes were all within the expected range of approximately 1.1-1.3 kb, indicating a 100% insertion rate.

[0140] 1.4.2 Sequencing: Positive clones were selected and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. Sequencing quality control results showed that the diversity of heavy and light chains in the library was relatively comprehensive, covering most of the major families of germline genes.

[0141] 1.5 Expression of MDA5 protein

[0142] The MDA5 gene sequence was artificially synthesized and recombined into the expression vector plasmid pFastBac1 to obtain the MDA5-pFastBac1 expression vector; the cloning site was EcoRI / Xba1, and the amino acid sequence of MDA5 was (SEQ ID NO:11):

[0143]

[0144] The gene sequence is (SEQ ID NO:12):

[0145]

[0146] The MDA5-pFastBac1 expression vector was transfected into DH10Bac competent cells and cultured. The precipitate was collected and subjected to GST tag affinity chromatography to obtain MDA5 protein. The purified MDA5 was then subjected to SDS-PAGE (polyacrylamide gel electrophoresis) to verify its purity. The SDS-PAGE electrophoresis image of the purified MDA5 is shown below. Figure 4 As shown, the protein size is 130 kDa and the purity is greater than 95%.

[0147] Example 2

[0148] This example demonstrates the preparation of MDA5 antibodies.

[0149] The reagents used are shown in Table 8.

[0150] Table 8

[0151]

[0152] 1. Biological panning

[0153] 1.1. Coating: Coat the immunotubes with 1 mL of MDA5 solution (50 μg / mL, CBS / PBS buffer) per tube and incubate overnight at 4°C.

[0154] 1.2. Washing: Discard the liquid in the immunoassay tube and wash three times with 5 mL of 0.05% PBST.

[0155] 1.3. Blocking: Add 5 mL of 5% skim milk (dissolved in PBST) to the tube and incubate at 30°C for 1 h.

[0156] 1.4. Washing: Discard the liquid in the immunoassay tube and wash once with 5 mL PBS.

[0157] 1.5. Negative screening: Add 0.5% skim milk (dissolved in PBST) to the phage library, add PBS to a total volume of 1 mL, and incubate at room temperature for 0.5 h.

[0158] 1.6. Incubation: Add 1 mL of negatively screened phage to each immunoassay tube and incubate at 30°C for 2 h.

[0159] 1.7. Washing: Discard the liquid in the immunoassay tube and wash six times with 5 mL of 0.05% PBST.

[0160] 1.8. Elution: Elute the phages bound to MDA5 with 1 mL of glycine-hydrochloric acid (pH=2.2), incubate with shaking at room temperature for about 7 min, and then neutralize the solution with 125 μL of Tris-HCl (pH=9.6) to pH=7.0~8.0.

[0161] 1.9. Determination: After eluting, the phage was diluted and used to infect logarithmic-phase Escherichia coli TG1. The titer was determined by plating.

[0162] 2. Amplification of eluted bacteriophages

[0163] 2.1. Take the eluted phage and add it to the logarithmic phase Escherichia coli TG1 bacterial culture. After standing at 37°C for 30 min, incubate at 220 rpm for 0.8 h.

[0164] 2.2. Add antibiotic Amp to the culture medium and incubate at 37℃ and 220 rpm until the bacterial OD of the culture solution is about 0.4~0.6.

[0165] 2.3. Add helper phages to the bacterial culture. Incubate at 37°C for 30 min, then incubate at 220 rpm for 50 min.

[0166] 2.4. Centrifuge the bacterial culture at 4000 rpm and discard the supernatant. Resuspend the bacterial cells in an equal volume of 2YT-Amp-Kan medium. Incubate overnight at 30°C and 220 rpm.

[0167] 2.5. The following day, centrifuge the bacterial culture at 8000 rpm, 4°C, for 20 min, and transfer the supernatant to a new centrifuge tube. Add 1 / 4 volume of PEG / NaCl solution. Mix thoroughly and incubate on ice or at 4°C for 1.5 h.

[0168] 2.6. Centrifuge at 8000 rpm, 4℃ for 30 min, and discard the supernatant. Resuspend the precipitate in approximately 1 mL of PBS. Centrifuge at 8000 rpm for 10 min, and transfer the supernatant to a new centrifuge tube.

[0169] 2.7. Dilute the amplified phage, infect TG1 cells in the logarithmic growth phase, and determine the titer by plating.

[0170] 3. Selection

[0171] The selection criteria are shown in Table 9.

[0172] Table 9

[0173]

[0174] The selection results are shown in Table 10.

[0175] Table 10

[0176]

[0177] 4. Polyclonal phage ELISA

[0178] 4.1. Coating: Coat the immunoassay plate and incubate overnight at 4°C. Experimental group (Ag): 100 μL / well MDA5 protein (4 μg / mL), control group 1 (NC1): 100 μL / well irrelevant protein (4 μg / mL), control group 2 (NC2): 100 μL / well PBS (0 μg / mL).

[0179] 4.2. Washing: Discard the liquid in the plate and wash each well three times with 300 μL of 0.05% PBST.

[0180] 4.3. Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 h.

[0181] 4.4. Incubation: Dilute the amplified phage with PBS after each round of amplification, increasing the dilution factor by 3-fold. The initial concentration is 3 × 10⁻⁶. 11 pfu / mL. Add 100 μL of diluted amplified phage to each well. Incubate at 32°C for 1 h.

[0182] 4.5. Washing: Same as step 4.2.

[0183] 4.6. Incubation: Add 100 μL of anti-M13-HRP antibody (1:9000) diluted with blocking buffer to each well and incubate at 30°C for 1 h.

[0184] 4.7. Washing: Same as step 4.2.

[0185] 4.8. Color development: Add 100 μL TMB to each well and develop color at room temperature in the dark for 5 min. Then add 50 μL 2 M HCl to each well to terminate the reaction.

[0186] 4.9. Plate reading: Use an ELISA reader to read the values ​​at 450 nm.

[0187] The results of polyclonal phage ELISA are shown in Table 11. The results show that as the number of panning rounds increases, the difference between the experimental group and the control group becomes larger and larger, indicating that enrichment occurs.

[0188] Table 11

[0189]

[0190] 5. Monoclonal phage ELISA

[0191] 5.1. Select the third round of eluted phage, dilute it to an appropriate concentration, infect TG1 cells in the logarithmic phase, and plate them.

[0192] 5.2. The next day, 96 single clones were picked from the plate and inoculated into a 96-well plate. The plate was then cultured at 37°C with shaking at 250 rpm until the bacterial OD value reached 0.4-0.6.

[0193] 5.3. Add helper phages to the 96-well plate medium. Incubate at 37°C for 30 min, then shake at 250 rpm at 37°C for 1 h.

[0194] 5.4. Centrifuge the 96-well plate at 4000 rpm for 5 min and discard the supernatant. Resuspend the bacterial culture in 2 YT-Amp-Kan medium in each well and incubate overnight at 30°C with shaking at 250 rpm.

[0195] 5.5. The next day, the 96-well plate was centrifuged at 4000 rpm for 13 min, and the supernatant was used for ELISA experiments.

[0196] 5.6. Coating: Coat the immunoassay plate and incubate overnight at 4°C. Antigen group (Ag): 100 μL / well MDA5 protein (4 μg / mL), control group (NC): 100 μL / well irrelevant protein (4 μg / mL).

[0197] 5.7. Washing: Discard the liquid in the plate and wash each well three times with 300 μL of 0.05% PBST.

[0198] 5.8. Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 1 h.

[0199] 5.9. Incubation: Add 100 μL of phage supernatant to each well and incubate at 32°C for 1 h.

[0200] 5.10. Washing: Same as step 5.7.

[0201] 5.11. Incubation: Add 100 μL of anti-M13-HRP antibody (1:9000) diluted with blocking buffer to each well and incubate at 32°C for 1 h.

[0202] 5.12. Washing: Same as step 5.7.

[0203] 5.13. Color development: Add 100 μL TMB to each well and develop color at room temperature in the dark for 6 min. Then add 50 μL 2M HCl to each well to terminate the reaction.

[0204] 5.14. Plate reading: Use an ELISA reader to read the values ​​at 450 nm.

[0205] The results of the antigen group monoclonal phage ELISA are shown in Table 12, and the results of the control group monoclonal phage ELISA are shown in Table 13. After verification, there was a significant difference between the antigen group and the control group, indicating that highly specific positive clones were screened.

[0206] Table 12

[0207]

[0208] Table 13

[0209]

[0210] 6. Secondary verification of positive clones

[0211] 6.1. Take 50 μL of positive clones and inoculate them into 2 mL of 2YT-AG medium, and culture until OD600 = 0.4~0.6.

[0212] 6.2. Add helper phages to the culture medium. Incubate at 37°C for 30 min, then shake at 250 rpm at 37°C for 45 min.

[0213] 6.3. Centrifuge the bacterial culture at 4000 rpm for 5 min and discard the supernatant. Resuspend the bacterial culture in 2 mL of 2YT-Amp-Kan medium in each well and incubate overnight at 30°C with shaking at 250 rpm.

[0214] 6.4. Centrifuge the overnight culture at 5000 rpm for 5 min, and take the supernatant for ELISA.

[0215] 6.5. Coating: Coat the immunoassay plate and incubate at 32°C for 2 h. Experimental group (Ag): 100 μL / well MDA5 protein (4 μg / mL), control group 1 (NC1): 100 μL / well irrelevant protein (4 μg / mL), control group 2 (NC2): 100 μL / well PBS (0 μg / mL).

[0216] 6.6. Washing: Discard the liquid in the plate and wash each well three times with 300 μL of 0.05% PBST.

[0217] 6.7. Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 h.

[0218] 6.8. Incubation: Add 100 μL of phage supernatant (5-fold serial dilution) to each well and incubate at 32°C for 2 h.

[0219] 6.9. Washing: Same as step 6.6.

[0220] 6.10. Incubation: Add 100 μL of anti-M13-HRP antibody (1:9000) diluted with blocking buffer to each well and incubate at 32°C for 1 h.

[0221] 6.11. Washing: Same as step 6.6.

[0222] 6.12. Color development: Add 100 μL TMB to each well and develop color at room temperature in the dark for 6 min. Then add 50 μL 2M HCl to each well to terminate the reaction.

[0223] 6.13. Plate reading: Use an ELISA reader to read values ​​at 450 nm and sequence highly specific clones.

[0224] The results of the secondary ELISA of the positive clones are shown in Table 14. The data of the antigen group and the control group of the MDA5-R3P1-E1 positive clone showed significant differences, indicating that the antibody has high specificity.

[0225] Table 14

[0226]

[0227] The positive clones were sequenced, and the nucleic acid sequence of the obtained MDA5 high-specificity antibody (named MDA5-R3P1-E1) is shown in SEQ ID NO:1, the heavy chain amino acid sequence is shown in SEQ ID NO:2, and the light chain amino acid sequence is shown in SEQ ID NO:3. Specifically, the heavy chain CDR1-CDR3 amino acid sequences are shown in SEQ ID NO:6-SEQ ID NO:8, and the light chain CDR1-CDR3 amino acid sequences are shown in SEQ ID NO:9, DNN, and SEQ ID NO:10, respectively.

[0228] SEQ ID NO:1:

[0229] .

[0230] Example 3

[0231] This embodiment demonstrates the use of ELISA to detect the OD values ​​of antibodies at different dilution concentrations.

[0232] Enzyme-linked immunosorbent assay (ELISA) experimental procedures:

[0233] 1. Coating: Coat the microplate with 100 μL of MDA5 protein per well (5 μg / mL) and incubate overnight at 4°C.

[0234] 2. Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0235] 3. Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 h.

[0236] 4. Positive antibody incubation: The MDA5-R3P1-E1 antibody was serially diluted, and 100 μL of the diluted antibody solution was added to each well. The mixture was incubated at 37°C for 1 h.

[0237] 5. Washing: Same as step 2.

[0238] 6. Secondary antibody incubation: Dilute Goat Anti-Human IgG (H+L) antibody (Jackson, code: 109-035-088) 10000 times with blocking buffer, add 100 μL of diluted secondary antibody to each well, and incubate at 37℃ for 30 min.

[0239] 7. Washing: Same as step 2.

[0240] 8. Color development: Add 100 μL TMB to each well, incubate at 37℃ for 10 min, and then add 50 μL 2M HCl to each well to terminate the reaction.

[0241] 9. Plate reading: Use a microplate reader to read the values ​​at 450 nm. The results are as follows: Figure 5 As shown, the results indicate that the MDA5-R3P1-E1 antibody has good binding affinity to the antigen.

[0242] Example 4

[0243] This embodiment uses Western blotting to validate the MDA5-R3P1-E1 antibody.

[0244] Western blot experimental procedure:

[0245] 1. Preparation of the lower layer adhesive: Mix 4 mL of 7.5% lower layer adhesive solution, 4 mL of 7.5% lower layer adhesive buffer and 80 μL of coagulant, pour the mixture into an adhesive slab, press the adhesive with 1.5 mL of anhydrous ethanol, wait for solidification, and then recover the anhydrous ethanol.

[0246] 2. Preparation of the top layer adhesive: Continue to pour the adhesive sheet with 1 mL of top layer adhesive solution, 1 mL of top layer adhesive buffer and 20 μL of accelerator, and wait for the adhesive sheet to solidify after 30 min.

[0247] 3. Electrophoresis: Add 100 μg of MDA5 protein to each well. After loading the sample, adjust the voltage to 80 V to start electrophoresis. Once the marker (M, protein molecular weight standard) is complete, adjust the voltage to 120 V. Stop electrophoresis when the marker reaches the end of the gel.

[0248] 4. Transfer: Place three layers of filter paper on a sponge, carefully place the gel on top, and then place a membrane of the same size as the gel on top, removing air bubbles. Stack two layers of filter paper and one layer of sponge, clamp the sandwich tightly, and place it in the electrophoresis tank. Add transfer buffer, and place the electrophoresis tank in a foam box, surrounding it with ice. Wet transfer at 300 A for 120 min.

[0249] 5. Blocking: Block with 5% skim milk (dissolved in TPBS) for 1 h.

[0250] 6. Primary antibody incubation: Dilute MDA5-R3P1-E1 antibody to 1 μg / mL with antibody dilution buffer and incubate overnight at 4°C.

[0251] 7. Washing the membrane: Wash three times with 1×TBST, 10 minutes each time.

[0252] 8. Secondary antibody incubation: Dilute Goat Anti-Human IgG (H+L) antibody (Jackson, code: 109-035-088) 20,000 times with antibody dilution buffer and incubate at 37°C for 1 h.

[0253] 9. Wash the film: Same as step 7.

[0254] 10. Luminescence detection imaging.

[0255] The results are as follows Figure 6 As shown, the antibody binds strongly to the recombinant protein, indicating that the antibody can be used for protein immunoblotting experiments.

[0256] In summary, this invention reverse transcribes RNA from MDA5 antibody-positive patients into cDNA, amplifies the VH and VL fragments of the DNA, further constructs an MDA5 antibody phage display library, identifies positive clones, and verifies polyclonal phages using ELISA. Based on the polyclonal results, a fourth round of single-clone screening is conducted, selecting 96 clones for single-clone ELISA testing of the antigen group and control group. Specific clones selected through single-clone screening are sequenced to eliminate erroneous and duplicate antibody sequences. Combined with the antigen-antibody specific binding ability reflected in the ELISA experiment, a fully humanized high-affinity MDA5 antibody, named MDA5-R3P1-E1, is finally obtained. ELISA and Western blotting verification show that this antibody has high activity, good stability, and strong specificity, making it a reference standard for qualitative detection of MDA5 positivity and also suitable for quantitative detection of anti-MDA5 autoantibody levels in patients with amyopathy dermatomyositis.

[0257] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A humanized monoclonal antibody against MDA5, characterized in that, The amino acid sequence of the heavy chain CDR1 of the antibody is shown in SEQ ID NO:6, the amino acid sequence of CDR2 is shown in SEQ ID NO:7, the amino acid sequence of CDR3 is shown in SEQ ID NO:8, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO:9, the amino acid sequence of CDR2 is DNN, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

10.

2. The MDA5 humanized monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

5.

3. The MDA5 humanized monoclonal antibody according to claim 2, characterized in that, The heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:2, and the light chain amino acid sequence is shown in SEQ ID NO:

3.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the MDA5 humanized monoclonal antibody as described in any one of claims 1-3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule as described in claim 4.

6. An engineered cell, characterized in that, The engineered cells contain the nucleic acid molecules as described in claim 4.

7. The method for preparing the humanized monoclonal antibody MDA5 according to any one of claims 1-3, characterized in that, The preparation method includes: The nucleic acid sequence encoding the humanized monoclonal antibody of MDA5 according to any one of claims 1-3 is transduced into a host cell and expressed, and the humanized monoclonal antibody of MDA5 is obtained by purification.

8. The use of the MDA5 humanized monoclonal antibody according to any one of claims 1-3 in any one of the following (1)-(5): (1) Prepare reagents or kits for detecting MDA5; (2) Prepare reagents or kits for diagnosing MDA5 antibody-positive dermatomyositis; (3) Detection of MDA5 for non-diagnostic and non-therapeutic purposes; (4) Prepare quality control materials for MDA5 antibody detection; (5) Preparation of a composition for MDA5 tracer.

9. A reagent or kit, characterized in that, The reagent or kit comprises the MDA5 humanized monoclonal antibody as described in any one of claims 1-3.

10. A method for detecting MDA5 for non-diagnostic and non-therapeutic purposes, characterized in that, The method includes detecting the sample to be tested using the MDA5 humanized monoclonal antibody according to any one of claims 1-3.

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