Human antibodies to dermatomyositis-specific antigen peptides, methods of making and uses thereof

Abstract

The present disclosure relates to human antibodies specific to dermatomyositis antigen peptides, preparation methods and uses. Specifically, the present disclosure relates to a monoclonal antibody that specifically binds to MDA5 antigen protein, a preparation method of the monoclonal antibody and a use of the aforementioned monoclonal antibody in the preparation of a drug for the diagnosis, prevention or treatment of inflammatory myopathy or its complications. The antibody or antigen-binding fragment against MDA5 screened by the present disclosure specifically binds to MDA5 antigen, can be used as a reference standard for qualitative detection of MDA5 positivity, realizes qualitative or quantitative detection of the level of anti-MDA5 autoantibody in patients with inflammatory myopathy or its complications, and has important significance for the clinical diagnosis, disease monitoring and treatment of patients with DM, CAMD, DM-ILD, CADM-ILD and the like.

Description

[0001] Priority and related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 202210259743.5, filed on March 16, 2022, entitled “Human Antibody of Dermatomyositis-Specific Antigenic Peptide, Preparation Method and Use”, the entire contents of which, including its appendices, are incorporated herein by reference. Technical Field

[0003] This disclosure pertains to the field of biomedicine and relates to a human antibody against a dermatomyositis-specific antigenic peptide, its preparation method, and its uses. Specifically, this disclosure relates to a monoclonal antibody that specifically binds to the MDA5 antigen protein, a method for preparing the monoclonal antibody, and its use in the preparation of medicaments for the diagnosis, prevention, or treatment of inflammatory myopathy or its complications. Background Technology

[0004] Inflammatory myopathy (IM) is a heterogeneous group of diseases characterized by inflammatory cell infiltration and myofiber necrosis in skeletal muscle. Dermatomyositis (DM) is the most common subtype of IM, mainly manifesting as varying degrees of skin, muscle, and lung involvement. The incidence of DM in adults is (1-6) / 100,000. A common complication of DM is interstitial lung disease (ILD), with an incidence of 23.1%-65%. Clinically, a type of DM without obvious muscle involvement is observed, termed clinically amypathic dermatomyositis (CADM). When CADM patients are complicated by acute interstitial pneumonia (AIP), the disease is characterized by rapid onset, rapid progression, and high mortality, with a mortality rate as high as 50% within 6 months.

[0005] Since the 1970s, myositis-specific autoantibodies (MSAs) have been increasingly detected in the serum of patients with melanoma (IM). Major antibodies in DM include anti-Mi-2 antibody, anti-transcriptional mediator 1-γ antibody, anti-MDA5 antibody, and anti-matrix protein 2 antibody. MDA5 is an intracellular sensor of viral RNA (including coronaviruses), encoding a protein of melanoma differentiation-related genes 5 (MDA5), which can trigger an innate immune response. In the early 21st century, CADM-140 antibody was screened from serum antibodies of patients with connective tissue diseases and idiopathic ILD; the antigen recognized by this antibody is the MDA5 protein. Anti-MDA5 antibodies are present in the serum of 35% of DM patients and 75% of CADM patients, and the incidence of rapidly progressive ILD is significantly higher in antibody-positive patients than in antibody-negative patients (50% vs. 6%, P = 0.008).

[0006] Studies have shown that anti-MDA5 antibody levels are closely related to the severity of skin ulcers, the severity of interstitial lung disease (ILD), treatment, and prognosis in patients with diabetes mellitus (DM) and cardiac arteriovenous dermatitis (CADM). In patients with anti-MDA5 antibody levels ≥500 U / mL, ulcers are multiple and deep-seated, while those in patients with antibody levels <500 U / mL are more superficial and solitary. Compared to anti-MDA5 antibody-negative patients (AIP incidence approximately 20%), anti-MDA5 antibody-positive DM and CADM patients have a higher AIP incidence (4%-33%), suggesting that anti-MDA5 antibody is a serological marker for AIP. The incidence of ILD in anti-MDA5 antibody-positive DM and CADM patients (42%-100%) is significantly higher than in anti-MDA5 antibody-negative patients (10%-30%), and most patients with ILD exhibit a rapidly progressive or subacute clinical pattern, dying from ILD and its complications within a short period (within 6 months). Furthermore, patients with high-titer anti-MDA5 antibodies in CADM-ILD have a poorer prognosis, and their anti-MDA5 antibody titers significantly decrease or even disappear after immunotherapy. Therefore, quantitative detection of anti-MDA5 antibodies is not only beneficial for the early assessment of ILD progression but also significant for monitoring disease activity and evaluating treatment efficacy.

[0007] In China, the current methods for detecting anti-MDA5 antibodies are all qualitative, namely Western blotting and immunostrip methods. These methods result in poor consistency and cannot quantitatively detect the level of anti-MDA5 antibodies in patient serum. Therefore, how to rapidly and accurately diagnose this type of disease and quantitatively detect the level of anti-MDA5 antibodies in patient serum is a crucial problem that urgently needs to be solved.

[0008] Humanized antibodies represent a major direction in the development of therapeutic antibodies, and the emergence of phage display technology has provided an excellent technical platform for their preparation, gradually becoming one of the main methods for obtaining humanized antibodies. Phage display technology, first established by Smith in 1985, has been widely applied in antigen-antibody library construction, drug design, vaccine research, pathogen detection, gene therapy, antigen epitope research, and cell signal transduction research after more than 30 years of development and refinement. Phage display technology utilizes polymerase chain reaction (PCR) to amplify the complete variable region genes of antibodies. Through phage surface display technology, Fab fragments or single-chain antibodies (ScFv) are expressed on the surface of phages, thereby screening and enriching specific antibodies. It has been proposed that almost all recombinant human monoclonal antibodies that specifically react with antigens can be screened from single-pot antibody library systems. Therefore, when using phage antibody technology, various antibody fragments applicable to in vivo diagnosis or treatment can be obtained. Summary of the Invention

[0009] The problem the invention aims to solve

[0010] Based on the problems existing in the prior art, this disclosure identifies a human monoclonal antibody against the anti-dermatomyositis specific antigen MDA5.

[0011] Solution for solving the problem

[0012] (1) An isolated anti-MDA5 antibody or its antigen-binding fragment, comprising a light chain variable region, wherein the light chain variable region comprises one or more of the sequences shown below:

[0013] (a1) The amino acid sequence as shown in SEQ ID NO:3;

[0014] (a2) Compared with the sequence shown in SEQ ID NO:3, there are 1, 2 or 3 conserved amino acid sequences;

[0015] (a3) The amino acid sequence shown in SEQ ID NO:4;

[0016] (a4) Compared with the sequence shown in SEQ ID NO:4, there is one or two conserved mutated amino acid sequences;

[0017] (a5) The amino acid sequence as shown in SEQ ID NO:5;

[0018] (a6) Compared with the sequence shown in SEQ ID NO:5, there are 1, 2 or 3 conserved amino acid sequences;

[0019] (a7) The amino acid sequence as shown in SEQ ID NO:41;

[0020] (a8) Compared with the sequence shown in SEQ ID NO:41, there are 1, 2 or 3 conserved amino acid sequences;

[0021] (a9) The amino acid sequence as shown in sequence KVS;

[0022] (a10) Compared with the sequence shown in KVS, there is one or two conserved amino acid mutations;

[0023] (a11) The amino acid sequence as shown in SEQ ID NO:43;

[0024] (a12) Compared with the sequence shown in SEQ ID NO:43, there are 1, 2 or 3 conserved amino acid sequences;

[0025] The variable region of the light chain is encoded according to the analysis method of IMGT.

[0026] (2) The antibody or antigen-binding fragment thereof according to (1) comprises a heavy chain variable region, wherein the heavy chain variable region comprises one or more of the following sequences:

[0027] (b1) The amino acid sequence as shown in SEQ ID NO:6;

[0028] (b2) Compared with the sequence shown in SEQ ID NO:6, there are 1, 2 or 3 conserved amino acid sequences;

[0029] (b3) The amino acid sequence as shown in SEQ ID NO:7;

[0030] (b4) Compared with the sequence shown in SEQ ID NO:7, there are 1, 2 or 3 conserved amino acid sequences;

[0031] (b5) The amino acid sequence as shown in SEQ ID NO:8;

[0032] (b6) Compared with the sequence shown in SEQ ID NO:8, there are 1, 2 or 3 conserved amino acid sequences;

[0033] (b7) The amino acid sequence as shown in SEQ ID NO:44;

[0034] (b8) Compared with the sequence shown in SEQ ID NO:44, there are 1, 2 or 3 conserved amino acid sequences;

[0035] (b9) The amino acid sequence as shown in SEQ ID NO:45;

[0036] (b10) Compared with the sequence shown in SEQ ID NO:45, there are 1, 2 or 3 conserved amino acid sequences;

[0037] (b11) The amino acid sequence as shown in SEQ ID NO:46;

[0038] (b12) Compared with the sequence shown in SEQ ID NO:46, there are 1, 2 or 3 conserved amino acid sequences;

[0039] The heavy chain variable region is encoded according to the IMGT analysis method.

[0040] (3) The antibody or its antigen-binding fragment according to (1) or (2) comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; and,

[0041] The LCDR1 contains an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:41, the LCDR2 contains an amino acid sequence as shown in SEQ ID NO:4 or sequence KVS, and the LCDR3 contains an amino acid sequence as shown in SEQ ID NO:5 or SEQ ID NO:43.

[0042] The HCDR1 contains an amino acid sequence as shown in SEQ ID NO:6 or SEQ ID NO:44, the HCDR2 contains an amino acid sequence as shown in SEQ ID NO:7 or SEQ ID NO:45, and the HCDR3 contains an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:46.

[0043] (4) The antibody or antigen-binding fragment thereof according to any one of (1)-(3), wherein the antibody or antigen-binding fragment thereof comprises one or more of the sequences shown below:

[0044] (i) The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:22 or SEQ ID NO:59, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:21 or SEQ ID NO:42;

[0045] (ii) Sequences with conserved mutations compared to the sequence shown in (i);

[0046] Preferably, the antibody or its antigen-binding fragment comprises one or more of the following sequences:

[0047] (iii) The heavy chain contains an amino acid sequence as shown in SEQ ID NO:18 or SEQ ID NO:56, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:20 or SEQ ID NO:58;

[0048] (iv) is a sequence with conserved mutations compared to the sequence shown in (iii).

[0049] (5) A polynucleotide, wherein the polynucleotide is selected from any one of (a)-(d):

[0050] (a) A nucleotide sequence comprising any one or a combination of sequences such as SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:55, SEQ ID NO:57;

[0051] (b) A nucleotide sequence comprising the reverse complementary sequence of the nucleotide sequence shown in any one or a combination of SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:55, SEQ ID NO:57;

[0052] (c) The reverse complementary sequence of a sequence that can hybridize with any of the nucleotide sequences shown in (a)-(b) under high-strict hybridization conditions or very high-strict hybridization conditions;

[0053] (d) A sequence that has at least 90% sequence identity with any of the nucleotide sequences shown in (a)-(c).

[0054] (6) A vector, wherein the vector comprises the polynucleotide according to (5).

[0055] (7) An isolated host cell, wherein the host cell comprises a vector as described in (6).

[0056] (8) A method for preparing host cells that stably express a target protein, wherein the method comprises the step of transforming an initial host cell using the vector described in (6).

[0057] (9) A method for preparing a target protein, the method comprising preparing the target protein using the host cell described in (7) or by the method described in (8).

[0058] (10) An antibody or its binding fragment prepared according to the method described in (9).

[0059] (11) A method for detecting anti-MDA5 antibody, wherein the method includes the step of detecting a sample to be tested using any one of (1)-(4) or the antibody or antigen-binding fragment thereof described in (10);

[0060] Optionally, the method includes a step of quantifying the anti-MDA5 antibody in the sample to be tested.

[0061] (12) A kit comprising an antibody or an antigen-binding fragment thereof according to any one of (1)-(4) or according to (10).

[0062] (13) A composition comprising an antibody or an antigen-binding fragment thereof according to any one of (1)-(4) or according to (10).

[0063] (14) Use of the antibody or antigen-binding fragment thereof according to any one of (1)-(4) or (10), or the composition according to (13) in at least one of (1)-(4):

[0064] (1) Detect anti-MDA5 antibodies, or prepare reagents or kits for detecting anti-MDA5 antibodies;

[0065] (2) Prepare reagents or kits for diagnosing inflammatory myopathy or its complications;

[0066] (3) Prepare reagents or kits for monitoring the progression of inflammatory myopathy or its complications;

[0067] (4) Prepare reagents or kits for studying the pathogenesis of inflammatory myopathy or its complications;

[0068] Optionally, the inflammatory myopathy is selected from at least one of the following: dermatomyositis DM, amyopathy-associated dermatomyositis CADM; alternatively, the complication is selected from at least one of the following: interstitial lung disease (ILD), acute interstitial pneumonia (AIP).

[0069] (15) A method for preventing or treating inflammatory myopathy or its complications, wherein the subject is given an antibody or antigen-binding fragment thereof according to any one of (1)-(4) or (10), or a composition according to (13);

[0070] Optionally, the inflammatory myopathy is selected from at least one of the following: dermatomyositis DM, amyopathy-associated dermatomyositis CADM; alternatively, the complication is selected from at least one of the following: interstitial lung disease (ILD), acute interstitial pneumonia (AIP).

[0071] The effects of the invention

[0072] This public screening yielded a humanized monoclonal antibody against the dermatomyositis-specific antigen MDA5. This antibody exhibits high activity, good stability, and strong specificity in binding to MDA5. By binding to MDA5, it can serve as a qualitative reference standard for detecting positive anti-MDA5 antibodies and can also quantitatively detect the level of anti-MDA5 autoantibodies in patients with inflammatory myopathy or its complications. This provides effective evidence for the diagnosis, disease monitoring, prognostic assessment, and disease mechanism research of patients with DM, CAMD, DM combined with ILD (also known as DM-ILD), and CAMD combined with ILD (also known as CADM-ILD), and can be used for the clinical diagnosis and treatment of IM and IM-ILD patients. Attached Figure Description

[0073] Figure 1 The image shows agarose gel electrophoresis image of a single clone of the VL phage library; where Lane M: DL2000, Lane 1-16: 21000076F pATA-VK, Lane 17-32: 21000076F pATA-Vλ.

[0074] Figure 2 The image shows agarose gel electrophoresis image of a single clone of the KH phage library; where Lane M: DL2000, Lane 1-48: 21000076F pATA-scFv-KH.

[0075] Figure 3 The image shows a single-clone PCR agarose gel electrophoresis image of the λH phage library; where Lane M: DL2000, Lane 1-48: 21000076F pATA-scFv-λH.

[0076] Figure 4 The results of monoclonal sequencing analysis of the phage display library are shown; the left figure shows the light chain analysis results of the library sequence, and the right figure shows the heavy chain analysis results of the library sequence.

[0077] Figure 5 The SDS-PAGE electrophoresis image of recombinant protein MDA5 is shown; the protein size is 130 kDa and the purity is greater than 95%.

[0078] Figure 6 The results of ELISA at different concentrations of 76F-MDA5-R2P1-G5 antibody dilution are shown.

[0079] Figure 7 The results of Western blot analysis of the expression of the 76F-MDA5-R2P1-G5 antibody in A549 and RAW cells are shown.

[0080] Figure 8The results of ELISA at different concentrations of the 76F-MDA5-R2P1-E10 antibody dilution are shown.

[0081] Figure 9 The results of Western blot verification of phosphorylation of stat1 and stat2 by 76F-MDA5-R2P1-E10 antibody are shown.

[0082] Figure 10 The results of the in vivo pro-inflammatory effects of 76F-MDA5-R2P1-E10 are shown. Detailed Implementation

[0083] definition

[0084] In the claims and / or specification of this disclosure, the words “a”, “an”, or “the” may mean “one”, but may also mean “one or more”, “at least one”, and “one or more”.

[0085] As used in the claims and specification, the words “comprising,” “having,” “including,” or “containing” mean inclusive or open-ended and do not exclude additional, uncited elements or method steps. At the same time, “comprising,” “having,” “including,” or “containing” can also mean closed-ended, excluding additional, uncited elements or method steps.

[0086] Throughout the application, the term “about” means: a value includes the standard deviation of the error of the apparatus or method used to determine that value.

[0087] While the disclosure supports the definition of the term "or" as merely a substitute and "and / or", the term "or" in the claims means "and / or" unless expressly stated as merely a substitute or as mutually exclusive among substitutes.

[0088] The term "inflammatory myopathy," also known as "IM," refers to a group of heterogeneous diseases characterized primarily by inflammatory cell infiltration and myofiber necrosis in skeletal muscle. "Inflammatory myopathy" and "IM" are used interchangeably.

[0089] The term "dermatomyositis" (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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] The terms “individual,” “patient,” or “subject” as used in the context of this invention include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0094] As used herein, the terms "MDA5," "MDA5 antigen," and "MDA5 protein" are used interchangeably. MDA5 has two N-terminal pattern recognition receptor (CRD) domains (caspase activation and recruitment domains) and one RNA helicase domain. The CRD 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.

[0095] As used in this disclosure, the term "conservative mutation" refers to a mutation (e.g., amino acid substitution, insertion, and / or deletion) that maintains the normal function of a protein. For example, a conservative mutation is a conservative substitution.

[0096] As used in this disclosure, a “conservative substitution” generally refers to the exchange of one amino acid at one or more sites in a protein. This substitution can be conserved. Examples of substitutions considered conserved include, specifically, substitutions of Ala to Ser or Thr, Arg to Gln, His, or Lys, Asn to Glu, Gln, Lys, His, or Asp, Asp to Asn, Glu, or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp, or Arg, Glu to Gly, Asn, Gln, Lys, or Asp, Gly to Pro, and His to Asn, Lys, Gln, Arg, or Tyr. Substitutions include: Ile to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val, or Phe; Phe to Trp, Tyr, Met, Ile, or Leu; Ser to Thr or Ala; Thr to Ser or Ala; Trp to Phe or Tyr; Tyr to His, Phe, or Trp; and Val to Met, Ile, or Leu. In addition, conserved mutations also include naturally occurring mutations arising from individual differences, strain differences, or species differences in gene origin.

[0097] In this disclosure, "sequence identity" and "identity percentage" refer to the percentage of identical (i.e., same) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotide or polypeptide and scoring the number of positions in the aligned polynucleotide or polypeptide containing the same nucleotide or amino acid residues, comparing this to the number of positions in the aligned polynucleotide or polypeptide containing different nucleotide or amino acid residues. Polynucleotides may differ at a position, for example, by containing different nucleotides (i.e., substitution or mutation) or deleted nucleotides (i.e., nucleotide insertion or deletion in one or two polynucleotides). Polypeptides may differ at a position, for example, by containing different amino acids (i.e., substitution or mutation) or deleted amino acids (i.e., amino acid insertion or deletion in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, the identity percentage can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0098] The term "phage display technology" in this disclosure refers to a biotechnology that inserts the DNA sequence of a foreign protein or polypeptide into an appropriate position in the structural gene of a phage coat protein, so that the foreign gene is expressed along with the expression of the coat protein, and the foreign protein is displayed on the surface of the phage as the phage is reassembled.

[0099] The term "antibody" as used herein in the broadest sense refers to a protein containing an antigen-binding site, encompassing a variety of natural and artificial antibodies, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, intact antibodies, and antibody fragments.

[0100] The term "antigen-binding fragment" in this disclosure refers to a portion or segment of a complete antibody with fewer amino acid residues than the complete antibody, capable of binding an antigen or competing with the complete antibody (i.e., the complete antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent or bispecific antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and bispecific or multispecific antibodies formed from antibody fragments.

[0101] The term "single-chain antibody" (scFv) in this disclosure refers to an antibody composed of a heavy chain variable region and a light chain variable region linked by a short peptide (also known as a linker) of a limited number of amino acids.

[0102] The term "Fab" fragment in this disclosure includes both a heavy chain variable domain and a light chain variable domain, and also includes a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues (including one or more cysteine ​​residues from the antibody hinge region) at the carboxyl terminus of the heavy chain CH1 domain. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments with a hinge cysteine ​​residue between them.

[0103] The term "complementarity-determining region" or "CDR region" or "CDR" refers to a region within the antibody variable domain that is sequence-hypervariant and forms a structurally defined loop ("hypervariant loop") and / or contains 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. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any or a combination of many known antibody CDR assignment systems, including, for example: Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (on the World Wide Web at imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0104] In the technical solutions described in this disclosure, unless otherwise specified, the antibody numbering scheme used for antibodies in this disclosure is the IMGT numbering scheme.

[0105] In some technical solutions, this disclosure relates to the stringency of hybridization conditions used to define the degree of complementarity between two polynucleotides. Optionally, the aforementioned polynucleotide may be selected from DNA. As used in this disclosure, “stringency” refers to the temperature and ionic strength conditions during hybridization and the presence of certain organic solvents. The higher the stringency, the greater the complementarity between the target nucleotide sequence and the labeled polynucleotide sequence. “Stringent conditions” refers to the temperature and ionic conditions under which only nucleotide sequences with high-frequency complementary bases will hybridize. The term “hybridization under high stringency or very high stringency conditions” as used herein describes the conditions used for hybridization and washing. Instructions for performing hybridization reactions can be found in Current Protocols in Molecule & Larbiology, John Wiley and Sons, NY (1989), 6.3.1–6.3.6. The specific hybridization conditions mentioned in this disclosure are as follows: 1) Highly stringent hybridization conditions: in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing once or more with 0.2X SSC and 0.1% SDS at 65°C; 2) Very highly stringent hybridization conditions: in 0.5M sodium phosphate and 7% SDS at 65°C, followed by washing once or more with 0.2X SSC and 1% SDS at 65°C.

[0106] Technical solution

[0107] In the technical solutions disclosed herein, the meanings of the nucleotide and amino acid sequence listing numbers are as follows:

[0108] The sequence shown in SEQ ID NO:1 is the amino acid sequence of the MDA5 antigen;

[0109] The sequence shown in SEQ ID NO:2 is a nucleotide sequence encoding the MDA5 antigen;

[0110] The sequence shown in SEQ ID NO:3 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody LCDR1;

[0111] The sequence shown in SEQ ID NO:4 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody LCDR2;

[0112] The sequence shown in SEQ ID NO:5 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody LCDR3;

[0113] The sequence shown in SEQ ID NO:6 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody HCDR1;

[0114] The sequence shown in SEQ ID NO:7 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody HCDR2;

[0115] The sequence shown in SEQ ID NO:8 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody HCDR3;

[0116] The sequence shown in SEQ ID NO:9 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody VL FR1;

[0117] The sequence shown in SEQ ID NO:10 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody VL FR2;

[0118] The sequence shown in SEQ ID NO:11 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody VL FR3;

[0119] The sequence shown in SEQ ID NO:12 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody VL FR4;

[0120] The sequence shown in SEQ ID NO:13 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody VH FR1;

[0121] The sequence shown in SEQ ID NO:14 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody VH FR2;

[0122] The sequence shown in SEQ ID NO:15 is the nucleotide sequence encoding the 76F-MDA5-R2P1-G5 antibody VH FR3;

[0123] The sequence shown in SEQ ID NO:16 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody VH FR4;

[0124] The sequence shown in SEQ ID NO:17 is a nucleotide sequence encoding the heavy chain of the 76F-MDA5-R2P1-G5 antibody;

[0125] The sequence shown in SEQ ID NO:18 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody heavy chain;

[0126] The sequence shown in SEQ ID NO:19 is a nucleotide sequence encoding the light chain of the 76F-MDA5-R2P1-G5 antibody;

[0127] The sequence shown in SEQ ID NO:20 is the amino acid sequence of the light chain of the 76F-MDA5-R2P1-G5 antibody;

[0128] The sequence shown in SEQ ID NO:21 is the amino acid sequence of antibody VL of 76F-MDA5-R2P1-G5;

[0129] The sequence shown in SEQ ID NO:22 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody VH;

[0130] The sequences shown in SEQ ID NO:23~40 are primer sequences.

[0131] The sequence shown in SEQ ID NO:41 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody LCDR1;

[0132] The sequence shown in KVS is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody LCDR2;

[0133] The sequence shown in SEQ ID NO:43 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody LCDR3;

[0134] The sequence shown in SEQ ID NO:44 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody HCDR1;

[0135] The sequence shown in SEQ ID NO:45 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody HCDR2;

[0136] The sequence shown in SEQ ID NO:46 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody HCDR3;

[0137] The sequence shown in SEQ ID NO:47 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody VL FR1;

[0138] The sequence shown in SEQ ID NO:48 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody VL FR2;

[0139] The sequence shown in SEQ ID NO:49 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody VL FR3;

[0140] The sequence shown in SEQ ID NO:50 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody VL FR4;

[0141] The sequence shown in SEQ ID NO:51 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody VH FR1;

[0142] The sequence shown in SEQ ID NO:52 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody VH FR2;

[0143] The sequence shown in SEQ ID NO:53 is the nucleotide sequence encoding the 76F-MDA5-R2P1-E10 antibody VH FR3;

[0144] The sequence shown in SEQ ID NO:54 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody VH FR4;

[0145] The sequence shown in SEQ ID NO:55 is a nucleotide sequence encoding the heavy chain of the 76F-MDA5-R2P1-E10 antibody;

[0146] The sequence shown in SEQ ID NO:56 is the amino acid sequence of the 76F-MDA5-R2P1-E10 antibody heavy chain;

[0147] The sequence shown in SEQ ID NO:57 is a nucleotide sequence encoding the light chain of the 76F-MDA5-R2P1-E10 antibody;

[0148] The sequence shown in SEQ ID NO:58 is the amino acid sequence of the light chain of the 76F-MDA5-R2P1-E10 antibody;

[0149] The sequence shown in SEQ ID NO:59 is the amino acid sequence of antibody VH of 76F-MDA5-R2P1-E10;

[0150] The sequence shown in SEQ ID NO:42 is the amino acid sequence of antibody VL of 76F-MDA5-R2P1-E10.

[0151] Anti-MDA5 antibody or its antigen-binding fragment

[0152] Anti-MDA5 antibodies are present in the serum of patients with diabetes mellitus (DM) and chronic arterial disease (CADM), and the incidence of rapidly progressive interstitial lung disease (ILD) is significantly higher in anti-MDA5 antibody-positive patients than in antibody-negative patients. MDA5 antibody-positive dermatomyositis is a rare but highly fatal autoimmune disease, with this subtype characterized by MDA5 antibody as a serological marker. Due to the rapid progression of lung disease, the six-month survival rate is only about 50%, often resulting in death from respiratory failure caused by ILD. Currently, the pathogenesis of MDA5 antibody-positive dermatomyositis is not fully understood, and the mainstream treatment regimen of combining hormones with traditional immunosuppressants is often ineffective and fails to significantly improve the survival rate of patients with MDA5 antibody-positive dermatomyositis complicated by rapidly progressive interstitial pneumonia. Classic hybridoma technology is a long-term, continuous experimental technique. Its problems lie in the lengthy preparation process and incomplete identification of antigenic epitopes, requiring subsequent humanization, making it difficult to obtain high-affinity human monoclonal antibodies and impossible to achieve absolute quantification of antibody levels. Therefore, these treatment methods lack specificity, have poor efficacy, and require long treatment courses.

[0153] Phage antibody library technology allows for the in vitro construction and screening of B cell antibody libraries from humans and other animals, bypassing steps such as immunization and cell fusion, thus shortening the experimental cycle and increasing stability. This enables the screening of fully human antibody sequences with high affinity in a short period. In this disclosure, PBMCs from MDA5 antibody-positive patients were isolated to construct a phage human antibody library, and human anti-MDA5 antigen monoclonal antibodies were screened. These antibodies, by specifically binding to MDA5, can quantitatively detect the level of anti-MDA5 autoantibodies in DM patients, thereby monitoring disease activity and aiding in the clinical diagnosis of DM. This invention provides a theoretical and experimental basis for specific treatment of DM and also offers a new research direction for treating autoimmune diseases with autoantibodies as the main pathogenic mechanism.

[0154] In some embodiments, this disclosure describes the preparation of the MDA5 protein. In some specific embodiments, the steps for preparing the MDA5 protein include:

[0155] (1) The artificially synthesized MDA5 domain gene was recombined into the expression vector plasmid pFastBac1 to obtain the MDA5-pFastBac1 expression vector.

[0156] (2) The MDA5-pFastBac1 expression vector was transfected into DH10Bac competent cells and cultured to obtain the MDA5 antigen protein.

[0157] In some embodiments, this disclosure utilizes a method for screening anti-MDA5 antibodies, which includes the following steps:

[0158] (1) A phage display library was constructed using PBMCs derived from patients who were positive for anti-MDA5 antibodies;

[0159] (2) Use the MDA5 antigen protein to screen the phage display library to obtain anti-MDA5 antibodies or their antigen-binding fragments that specifically bind to the anti-MDA5 antigen protein.

[0160] In some more specific implementations, methods for screening anti-MDA5 antibodies include:

[0161] PBMCs from MDA5 antibody-positive patients were isolated, and RNA was extracted and quality-checked. The quality-checked RNA was reverse-transcribed into cDNA using RT-PCR, and all VH and VL antibody gene fragments were amplified to form VK and Vλ libraries. 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 the plasmid vectors of the VK and Vλ libraries to form KH and λH libraries, respectively.

[0162] An antibody gene conjugate library was inserted immediately downstream of the leader sequence of gene III (g3) of a phage-encoded membrane protein. Through helper phage superinfection, the polypeptide or protein expressed by the exogenous antibody gene could be displayed as a fusion protein at the N-terminus of the phage coat protein pIII. Each phage particle encodes and presents a different antibody, containing billions of individual clones. In these antibody libraries, genes encoding antibodies that bind to antigens were selected through an in vitro affinity enrichment-mild elution-phage amplification process, repeated several times until a highly specific and affinity-rich antibody phage library was obtained. Positive clones were then screened from this library. Positive clones were identified using ELISA, and finally, highly specific and affinity-rich fully human antibodies were selected. The human monoclonal antibodies obtained from the screening were subjected to Western blotting with the human non-small cell lung cancer cell line A549 and the mouse peritoneal macrophage cell line RAW, using Gapdh and Actin as internal controls, respectively, to verify antibody efficacy.

[0163] This disclosure involves three rounds of screening of antibody phage libraries, identifying clones with an antigen group greater than 3 times that of the control group as positive clones, and performing sequencing analysis on these single clones. Erroneous and duplicate antibody sequences were eliminated, and combined with the antigen-antibody specific binding ability reflected in ELISA experiments, two high-affinity antibodies were finally obtained, named 76F-MDA5-R2P1-G5 and 76F-MDA5-R2P1-E10. ELISA and Western blotting validation showed that these antibodies have high activity, good stability, and strong specificity, and can serve as a reference standard for qualitative detection of MDA5 positivity, as well as for quantitative detection of anti-MDA5 autoantibody levels in patients with myopathic dermatomyositis (IM) and IM-ILD (e.g., DM, CADM, DM-ILD, CADM-ILD, etc.).

[0164] Example

[0165] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0166] All reagents used in the examples, unless otherwise emphasized, are commercially available.

[0167] Example 1: Method for constructing a human ScFv phage display library

[0168] Table 1. Main reagents used in this embodiment.

[0169]

[0170] 1. Library Construction

[0171] 1.1 Assemble the heavy chain variable region (VH) and the light chain variable region (VL)

[0172] Table 2. PCR reaction conditions and procedures

[0173]

[0174] The three steps of denaturation, annealing, and extension (1) are repeated 30 times.

[0175] Primer sequences:

[0176] Forward(F):

[0177] 5′L-VH 1:ACAGGTGCCCACTCCCAGGTGCAG (SEQ ID NO:23)

[0178] 5′L-VH 3:AAGGTGTCCAGTGTGARGTGCAG(SEQ ID NO:24)

[0179] 5′L-VH 4 / 6: CCCAGATGGGTCCTGTCCCAGGTGCAG(SEQ ID NO:25) 5′L-VH 5 / 7: CAAGGAGTCTGTTCCGAGGTGCAG(SEQ ID NO:26)

[0180] 5′L VK 1 / 2:ATGAGGSTCCCYGCTCAGCTGCTGG(SEQ ID NO:27)

[0181] 5′L VK 3:CTCTTCCTCCTGCTACTCTGGCTCCCAG(SEQ ID NO:28)5′L VK 4 / 5:ATTTCTCTGTTGCTCTGGATCTCTG(SEQ ID NO:29)

[0182] 5′L Vλ1:GGTCCTGGGCCCAGTCTGTGCTG(SEQ ID NO:30)

[0183] 5′L Vλ2:GGTCCTGGGCCCAGTCTGCCCTG(SEQ ID NO:31)

[0184] 5′L Vλ3:GCTCTGTGACCTCCTATGAGCTG(SEQ ID NO:32)

[0185] 5′L Vλ4 / 5:GGTCTCTCTCSCAGCYTGTGCTG(SEQ ID NO:33)

[0186] 5′L Vλ6:GTTCTTGGGCCAATTTTATGCTG(SEQ ID NO:34)

[0187] 5′L Vλ7:GGTCCAATTCYCAGGCTGTGGTG(SEQ ID NO:35)

[0188] 5′L Vλ8 / 9 / 10:GAGTGGATTCTCAGACTGTGGTG(SEQ ID NO:36)Reverse(R):

[0189] 3′CK:TGCTGTCCTTGCTGTCCTGCT(SEQ ID NO:37)

[0190] 3′Cλ:CACCAGTGTGGCCTTGTTGGCTTG (SEQ ID NO:38)

[0191] 1.2 Construction of a light chain variable region phage display library

[0192] 1.2.1 Preparing the pATA-scFv-2 vector for library cloning

[0193] 1.2.2 Digestion of vectors and PCR products

[0194] Table 3. Reaction system for digestion vector and PCR product

[0195]

[0196] 1.2.3 Connection

[0197] Table 4. Connection Reaction System

[0198]

[0199] Incubate overnight at 16°C, then inactivate by heating at 65°C for 10 minutes.

[0200] 1.2.4 Electrical Transfer

[0201] 1.2.4.1 Preparation of TG1 competent cells.

[0202] 1.2.4.2 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).

[0203] 1.2.4.3 Remove Electrocompetent cells from the freezer at -80°C and place them on ice until they are completely thawed (10-15 minutes). After thawing, gently mix the cells. Transfer 50 μL of cells to a frozen microcentrifuge tube placed on ice.

[0204] 1.2.4.4 Carefully add 3 μL of the DNA mixture to a frozen electroporation cuvette, being careful not to create air bubbles. Quickly flick the tube downwards with your wrist to allow the cells to settle at the bottom.

[0205] 1.2.4.5 Electroporation was performed at 600 Ω, 10 μF, and 1.8 kV. Within 10 seconds of the pulse, 1 mL of preheated SOC medium was immediately added to each tube. The tubes were incubated at 37°C with shaking at 250 rpm for 1 hour.

[0206] 1.2.4.6 Collect all electroporation media. Serially dilute 10 μL of culture into 90 μL of SOC medium and spread it onto LB / Amp / Glucose plates. 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 inoculum volume.

[0207] 1.3 Construction of VL-VH phage display library

[0208] 1.3.1 Digestion of vectors and PCR products

[0209] Table 5. Digestion reaction system

[0210]

[0211] 1.3.2 Connection

[0212] Table 6. Connection Reaction System

[0213]

[0214] Incubate overnight at 16°C, then inactivate by heating at 65°C for 10 minutes.

[0215] 1.3.3 Electrical Transfer

[0216] 1.3.3.1 Preparation of TG1 competent cells.

[0217] 1.3.3.2 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).

[0218] 1.3.3.3 Remove Electrocompetent cells from the freezer at -80°C and place them on ice until they are completely thawed (10-15 minutes). After thawing, gently mix the cells.

[0219] 1.3.3.4 Carefully add 6 μL of the DNA mixture to a frozen electroporation cuvette, being careful not to create air bubbles. Quickly flick the tube downwards with your wrist to allow the cells to settle at the bottom.

[0220] 1.3.3.5 Electroporation at 600 Ω, 10 μF, and 2.5 kV. Immediately within 10 seconds of the pulse, add 2 mL of preheated SOC medium to each tube. Incubate at 37°C with shaking at 250 rpm for 1 hour.

[0221] 1.3.3.6 Collect all electroporation media. Serially dilute 10 μL of culture into 90 μL of SOC medium and spread it onto LB / Amp / Glucose plates. 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 inoculum volume.

[0222] 1.4 Library Evaluation

[0223] 1.4.1 Colony PCR: PCR was performed using the constructed library as a template.

[0224] Table 7. PCR reaction conditions

[0225]

[0226] The three steps of denaturation, annealing, and extension (1) are repeated 30 times.

[0227] Primer sequences:

[0228] Forward(F):AGCGGATAACAATTTCACACAGGA(SEQ ID NO:39)

[0229] Forward(R):GCCCCCTTATTAGCGTTTTGCCATC(SEQ ID NO:40)

[0230] The results of agarose gel electrophoresis after PCR are as follows: Figures 1-3 As shown.

[0231] 1.4.2 Sequencing: Positive clones were selected and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing quality control results are as follows: Figure 4 As shown.

[0232] 1.5 Expression of MDA5 protein

[0233] 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 / XbaI.

[0234] The amino acid sequence of MDA5 (SEQ ID NO:1) is as follows:

[0235]

[0236] The gene sequence is shown in SEQ ID NO:2.

[0237] 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 5 As shown, the purity is greater than 95%.

[0238] Example 2: Preparation of monoclonal antibodies that specifically bind to MDA5

[0239] Table 8. Main reagents used in this embodiment

[0240] reagents serial number Manufacturer 96-well plate 42592 Costar Tween 20 P2287 Sigma Tris RES3098T-B7 Sigma Glycine G8200 Solarbio PEG 181986 Sigma PBS C10010500BT Life BSA A104912-100g aladdin Skim milk 6342932 BD

[0241] 1. First round

[0242] 1.1 Biological Screening

[0243] 1.1.1 Coating: Coat the immunotubes and incubate overnight at 4°C. Antigen group: 1 mL MDA5 transfection buffer (50 μg / mL), control group: 500 μL transfection buffer (0 μg / mL).

[0244] 1.1.2 Washing: Discard the liquid in the immunoassay tube and wash three times with 5 mL of 0.05% PBST.

[0245] 1.1.3 Blocking: Add 5 mL of 5% skim milk (dissolved in PBST) to the tube and incubate at 37°C for 2 hours.

[0246] 1.1.4 Washing: Discard the liquid in the immunoassay tube and wash once with 5 mL of 0.05% PBST.

[0247] 1.1.5 Incubation: Dilute the phage library with 1% skim milk (dissolved in PBST), add 1 mL to an immunoassay tube, and incubate at 32°C for 2 hours.

[0248] 1.1.6 Washing: Discard the liquid in the immunoassay tube, wash three times with 5 mL of 0.05% PBST, and wash twice with PBS.

[0249] 1.1.7 Elution: Elute the phages bound to MDA5 with 1 mL of glycine-hydrochloric acid (pH 2.2), and then neutralize to pH 7.0 with Tris-HCl.

[0250] 1.2 Determination of the titer of diluted bacteriophages

[0251] 1.2.1 Culture Escherichia coli TG1 until OD600 = 0.4-0.6.

[0252] 1.2.2 Mix 10 μL of diluted eluted phage with 190 μL of Escherichia coli TG1.

[0253] 1.2.3 Incubate the mixture at 37°C for 15 minutes, then pour it into 2×YT-A (Amp 100μg / mL) medium. Invert the medium and incubate overnight at 37°C.

[0254] 1.3 Phage Library Amplification

[0255] 1.3.1 Add 10 μL of E. coli TG1 to 800 μL of 2YT culture medium and mix and culture at 37℃ until OD600 = 0.4-0.6.

[0256] 1.3.2 Transfer the TG1 cultured to the logarithmic phase to 10 mL of 2YT-G culture medium (final concentration 2% glucose) and culture on a shaker at 37℃ until OD600 = 0.4-0.6.

[0257] 1.3.3 Add the eluted product, incubate at 37°C for 30 minutes, and then culture on a shaker at 37°C for 30 minutes.

[0258] 1.3.4 Add 30 mL of 2YT-AG culture medium (final concentration 0.1% Amp, 2% glucose) and incubate at 37°C in a shaker for 1 hour.

[0259] 1.3.5 Add M13KO7 (M13KO7:TG1 = 20:1), incubate at 37°C for 30 minutes, and then culture on a shaker at 37°C for 30 minutes.

[0260] 1.3.6 Centrifuge the bacterial culture at 5000 rpm for 5 minutes. Resuspend in 40 mL of 2YT-AK (final concentrations: Amp 100 μg / mL, Kan 100 μg / mL) and incubate overnight at 30°C on a shaker.

[0261] 1.3.7 Centrifuge at 8000 rpm for 10 minutes, remove the supernatant, resuspend in 1 mL PBS, centrifuge at 12000 rpm for 5 minutes, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0262] 1.4 Determination of titer of amplified phage library

[0263] The steps are the same as in 1.2.

[0264] 2. Rounds 2 to 3

[0265] 2.1 Biological Screening

[0266] Repeat step 1 twice, using eluted phage from the previous round of amplification for each phage library input.

[0267] Table 9. Results of biological screening

[0268]

[0269] 3. Polyclonal phage ELISA

[0270] 3.1 Coating: Coat the microplate and incubate overnight at 4°C. Antigen group: 100 μL MDA5 protein per well (4 μg / mL), control group: 100 μL protein dilution buffer per well (0 μg / mL).

[0271] 3.2 Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0272] 3.3 Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.

[0273] 3.4 Phage incubation: Add 100 μL of diluted phage to each well as shown in Table 10, and incubate at 32°C for 2 hours.

[0274] 3.5 Washing: Same as step 3.2.

[0275] 3.6 Secondary antibody 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 hour.

[0276] 3.7 Washing: Same as step 3.2.

[0277] 3.8 Color development: Add 100 μL TMB to each well, incubate at room temperature, and then add 50 μL 2M HCl to each well to terminate the reaction.

[0278] 3.9 Plate reading: Use an ELISA reader to read values ​​at 450nm-630nm.

[0279] Table 10. Results of polyclonal phage ELISA

[0280]

[0281] 4. Monoclonal phage ELISA (based on polyclonal results, the second round of elution products are used for monoclonal ELISA).

[0282] 4.1 Ninety-six clones were selected from the culture dish and cultured at 37°C and 250 rpm until OD was reached. 600 =0.4-0.6.

[0283] 4.2 M13KO7 infection culture (MOI = 20:1) was incubated at 37°C for 30 minutes, followed by shaking culture at 37°C for 30 minutes. The bacterial culture was centrifuged and the pellet was resuspended with an equal volume of 2×YT-AK (final concentrations of Amp 100 μg / mL and Kan 100 μg / mL), and incubated overnight at 30°C.

[0284] 4.3 Centrifuge the culture and use the supernatant for ELISA.

[0285] 4.4 Coating: Coat the microplate and incubate overnight at 4°C. Antigen group: 100 μL MDA5 protein per well (4 μg / mL), control group: 100 μL protein dilution buffer per well (0 μg / mL).

[0286] 4.5 Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0287] 4.6 Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.

[0288] 4.7 Phage incubation: Add 100 μL of phage supernatant to each well and incubate at 32°C for 2 hours.

[0289] 4.8 Washing: Same as step 4.5.

[0290] 4.9 Secondary antibody 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 hour.

[0291] 4.10 Washing: Same as step 4.5.

[0292] 4.11 Color development: Add 100 μL TMB to each well, incubate at room temperature, and then add 50 μL 2M HCl to each well to terminate the reaction.

[0293] 4.12 Plate reading: Use an ELISA reader to read values ​​at 450nm-630nm and sequence highly specific clones.

[0294] Table 11. Results of antigen-group monoclonal phage ELISA

[0295] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.04 0.04 0.04 0.04 0.04 0.03 0.03 0.90 0.10 0.05 0.13 0.03 B 0.06 0.07 0.08 0.07 0.03 0.05 1.20 0.03 0.03 0.10 1.72 0.10 C 0.04 0.26 0.03 0.03 0.05 0.15 0.03 0.15 0.05 0.04 0.07 0.30 D 0.05 0.04 0.04 0.04 0.04 0.04 1.19 0.22 0.21 0.05 0.21 0.08 E 0.04 0.04 0.27 0.03 0.03 0.03 0.23 0.03 0.04 0.43 0.05 0.55 F 0.08 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.05 0.03 0.04 0.04 G 0.04 0.04 0.05 0.27 1.68 0.03 0.37 0.03 0.04 0.03 0.04 0.08 H 0.05 0.04 0.03 0.07 0.66 0.23 0.04 0.05 0.04 0.03 0.04 0.05

[0296] Table 12. Results of monoclonal phage ELISA in the control group

[0297] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.04 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.05 0.16 B 0.02 0.02 0.02 0.03 0.02 0.02 0.11 0.03 0.02 0.02 0.05 0.07 C 0.03 0.02 0.23 0.03 0.02 0.04 0.04 0.03 0.03 0.02 0.03 0.12 D 0.03 0.02 0.03 0.03 0.03 0.03 0.04 0.03 0.02 0.03 0.03 0.12 E 0.03 0.02 0.03 0.02 0.03 0.03 0.02 0.33 0.02 0.03 0.03 0.10 F 0.04 0.02 0.02 0.02 0.03 0.02 0.02 0.03 0.02 0.02 0.03 0.04 G 0.06 0.02 0.02 0.05 0.08 0.02 0.06 0.16 0.03 0.03 0.13 0.04 H 0.11 0.05 0.03 0.06 0.10 0.23 0.04 0.11 0.04 0.04 0.04 0.12

[0298] 5. ELISA verification of positive clones

[0299] 5.1 Add 50 μL of positive clones to 2 mL of 2YT-AG medium (final concentration 0.1% Amp, 2% glucose) and incubate until OD600. 600 =0.4-0.6.

[0300] 5.2 M13KO7 infection culture (MOI = 20:1) was incubated at 37°C for 30 minutes, followed by shaking culture at 37°C for 30 minutes. The bacterial culture was centrifuged and the pellet was resuspended with an equal volume of 2×YT-AK (final concentrations of Amp 100 μg / mL and Kan 100 μg / mL), and incubated overnight at 30°C.

[0301] 5.3 Centrifuge the culture and use the supernatant for ELISA.

[0302] 5.4 Coating: Coat the microplate and incubate overnight at 4°C. Antigen group: 100 μL MDA5 protein per well (4 μg / mL), control group: 100 μL protein dilution buffer per well (0 μg / mL).

[0303] 5.5 Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0304] 5.6 Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.

[0305] 5.7 Phage incubation: Add 100 μL of phage supernatant to each well and incubate at 32°C for 2 hours.

[0306] 5.8 Washing: Same as step 4.5.

[0307] 5.9 Secondary antibody 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 hour.

[0308] 5.10 Washing: Same as step 4.5.

[0309] 5.11 Color development: Add 100 μL TMB to each well, incubate at room temperature, and then add 50 μL 2M HCl to each well to terminate the reaction.

[0310] 5.12 Plate reading: Use an ELISA reader to read values ​​at 450nm-630nm and sequence highly specific clones.

[0311] Table 13. Results of ELISA for positive monoclonal phages

[0312]

[0313] The above positive clones were sent for sequencing, and the sequences of the highly specific antibodies obtained are as follows:

[0314] Table 14.76F-MDA5-R2P1-G5 antibody sequence

[0315]

[0316] Table 15.76F-MDA5-R2P1-E10 antibody sequence

[0317]

[0318] 6. Sequencing of antibody sequences

[0319] The phage-positive clones obtained through screening were subjected to full-sequence sequencing to obtain the corresponding antibody heavy and light chains, as well as the full sequences, as shown below:

[0320] The heavy chain base sequence (SEQ ID NO:17) of the 76F-MDA5-R2P1-G5 antibody is as follows:

[0321]

[0322] The amino acid sequence of the heavy chain of the 76F-MDA5-R2P1-G5 antibody (SEQ ID NO: 18) is as follows:

[0323] MKHLWFFLLLVAAPRWVLSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHRRYAFDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0324] The base sequence of the light chain of the 76F-MDA5-R2P1-G5 antibody (SEQ ID NO:19) is: GAATTCGCCGCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCTCTGCTGCTGTGGATCTCCGGCGCCTACGGCCAGGCTGTGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGC AACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTATTGTAGCTCATATGTAGTCAGCTACACTTGGGTATTCGGCGGAGGCACCAAGGTGACCGTCCTCCGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC;

[0325] The amino acid sequence of the light chain of the 76F-MDA5-R2P1-G5 antibody (SEQ ID NO:20) is:

[0326] MVLQTQVFISLLLWISGAYGQAVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYVVSYTWVFGGGTKVTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0327] The base sequence of the heavy chain of the 76F-MDA5-R2P1-E10 antibody (SEQ ID NO: 55) is:

[0328]

[0329] The heavy chain amino acid sequence (SEQ ID NO: 56) of the 76F-MDA5-R2P1-E10 antibody is as follows:

[0330] MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGIINPSGGSTNYAQKFQGRVTLTRDASTSTVYMELSSLRSEDTAVYYCARVRREQLLAKYYYGMDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0331] The light chain base sequence (SEQ ID NO: 57) of the 76F-MDA5-R2P1-E10 antibody is as follows:

[0332] GAATTCGCCGCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCTCTGCTGCTGTGGATCTCCGGCGCCTACGGCGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTCCATACTAATGGAAACACCTACCTGACTTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTTATTTACAAGGTTTCTAGTCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGACGATGTTGGGTTTTATTACTGCATGCAAGGTACACAGAAGCCGAAGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0333] The amino acid sequence of the light chain of the 76F-MDA5-R2P1-E10 antibody (SEQ ID NO:58) is as follows:

[0334] MVLQTQVFISLLLWISGAYGDIVMTQSPLSLPVTLGQPASISCRSSQSLLHTNGNTYLTWFQQRPGQSPRRLIYKVSSRDSGVPDRFSGSGSGTDFTLKISRVEADDVGFYYCMQGTQK PKTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0335] Example 3: ELISA detection of OD values ​​of antibodies at different dilution concentrations

[0336] Enzyme-linked immunosorbent assay (ELISA) experimental procedures:

[0337] 1. Coating: Coat the microplate with 100 μL of MDA5 protein per well (4 μg / mL) and incubate overnight at 4°C.

[0338] 2. Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0339] 3. Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.

[0340] 4. Positive antibody incubation: The 76F-MDA5-R2P1-G5 and 76F-MDA5-R2P1-E10 antibodies were serially diluted, and 100 μL of the diluted antibody solution was added to each well. The mixture was incubated at 37°C for 1 hour.

[0341] 5. Washing: Same as step 4.5.

[0342] 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°C for 30 minutes.

[0343] 7. Washing: Same as step 4.5.

[0344] 8. Color development: Add 100 μL TMB to each well, incubate at 37°C for 10 minutes, and then add 50 μL 2M HCl to each well to terminate the reaction.

[0345] 9. Plate reading: Use an ELISA reader to read values ​​at 450nm-630nm, as shown below. Figure 6 and Figure 8As shown in the figure, the results indicate that 76F-MDA5-R2P1-G5, 76F-MDA5-R2P1-E10 and MDA5 all have strong specific binding ability.

[0346] Example 4: Western blot validation of 76F-MDA5-R2P1-G5 antibody

[0347] The RAW cells used in this embodiment were RAW264.7 cells purchased from the ATCC cell bank, product number: Cat#TIB-71; and A549 cells purchased from the ATCC cell bank, product number: Cat#CCL-185.

[0348] Western blot experimental procedure:

[0349] 1. Cell collection: A549 cells and RAW cells were cultured to the logarithmic growth phase, and the cell pellet was collected. An appropriate amount of lysis buffer was added to lyse the cells (phosphatase and protease inhibitor: RIPA lysis buffer = 1:100), and the cells were incubated on ice for 30 minutes.

[0350] 2. Protein extraction: Centrifuge the lysed cells at 12,000 rpm for 15 minutes at 4°C and retain the supernatant. Add 1 / 3 volume of 4× Protein SDS loading buffer to the supernatant, boil at 100°C for 10 minutes, and then store the sample at 4°C.

[0351] 3. 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 1-2 mL of anhydrous ethanol, wait for solidification, and then recover the anhydrous ethanol.

[0352] 4. 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 30 minutes for the adhesive sheet to solidify.

[0353] 5. Electrophoresis: Add 30 μL of protein to each well. After loading the sample, adjust the voltage to 80V to start electrophoresis. After the markers are aligned, adjust the voltage to 120V and stop electrophoresis when the markers reach the end of the gel.

[0354] 6. 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 300A for 120 minutes.

[0355] 7. Blocking: Block with 5% skim milk (dissolved in TPBS) for 1 hour.

[0356] 8. Primary antibody incubation: Dilute 76F-MDA5-R2P1-G5 antibody 1:100 with antibody dilution buffer and incubate overnight at 4°C.

[0357] 9. Washing the membrane: Wash three times with 1XTBST, 10 minutes each time.

[0358] 10. 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 hour.

[0359] 11. Wash the film: Same as step 9.

[0360] 12. Luminescence detection imaging. For example... Figure 7 As shown in the figure. The results showed that 76F-MDA5-R2P1-G5 was expressed in both A549 and RAW cells.

[0361] Example 5: Western blot validation of the 76F-MDA5-R2P1-E10 antibody

[0362] Western blot experimental procedure:

[0363] 1. Cell stimulation: RAW cells (RAW264.7) were stimulated for 24 hours with Human IgG1 isotype control (4ug), poly(I:C) (4ug), MDA5 (1ug), 76F-MDA5-R2P1-E10 (4ug), and MDA5 (1ug) + 76F-MDA5-R2P1-E10 (4ug).

[0364] 2. Cell collection: After 24 hours, collect the cell pellet, add an appropriate amount of lysis buffer to lyse the cells (phosphatase and protease inhibitor: RIPA lysis buffer = 1:100), and incubate on ice for 30 minutes.

[0365] 3. Protein extraction: Centrifuge the lysed cells at 12,000 rpm for 15 minutes at 4°C and retain the supernatant. Add 1 / 3 volume of 4× Protein SDS loading buffer to the supernatant, boil at 100°C for 10 minutes, and then store the sample at 4°C.

[0366] 4. 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 with 1-2 mL of anhydrous ethanol, wait for solidification, and then recover the anhydrous ethanol.

[0367] 5. 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 30 minutes for the adhesive sheet to solidify.

[0368] 6. Electrophoresis: Add 30 μL of protein to each well. After loading the sample, adjust the voltage to 80V to start electrophoresis. After the markers are aligned, adjust the voltage to 120V and stop electrophoresis when the markers reach the end of the gel.

[0369] 7. 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 300A for 120 minutes.

[0370] 8. Blocking: Block with 5% skim milk (dissolved in TPBS) for 1 hour.

[0371] 9. Primary antibody incubation: Dilute p-stat1 (CST#8826) and p-stat2 (CST#88410) antibodies 1:100 with antibody dilution buffer and incubate overnight at 4°C.

[0372] 10. Washing the membrane: Wash three times with 1XTBST, 10 minutes each time.

[0373] 11. Secondary antibody incubation: Dilute HRP Goat Anti-Mouse IgG (H+L) (ABclonal, AS003) 10,000 times with antibody dilution buffer and incubate at 37°C for 1 hour.

[0374] 12. Wash the film: Same as step 10.

[0375] luminescence detection imaging, such as Figure 9 As shown in the figure, the results indicate that the 76F-MDA5-R2P1-E10 antibody can stimulate the phosphorylation of stat1 and stat2.

[0376] Example 6: Animal experiments to verify the in vivo pro-inflammatory effect of 76F-MDA5-R2P1-E10

[0377] Animal experimental procedures:

[0378] 1. Select 6-8 week old C57 WT female mice and administer 76F-MDA5-R2P1-E10 (3mg / kg) intraperitoneally on days 0, 2 and 4. The control group was treated with the same dose of Human IgG1 isotype control. Lung specimens were collected after euthanizing the mice on day 18.

[0379] 2. Mouse lung specimens were fixed with 4% PFA for 24 hours.

[0380] 3. Dehydration and clearing: The tissue was placed in different concentrations of alcohol for dehydration (75% ALC for 25 min, 85% ALC for 25 min, 2X95% ALC for 25 min, 2X100% ALC for 25 min); then the tissue block was placed in xylene, a clearing agent, for clearing.

[0381] 4. Paraffin embedding: Place the transparent tissue block in molten paraffin and keep it in a paraffin bath for insulation. After the paraffin has completely penetrated the tissue block, embed it.

[0382] 5. Slicing: Fix the embedded wax block onto a microtome and slice it into thin slices.

[0383] 6. HE staining: Before staining, remove paraffin from the sections with xylene, then pass through high-concentration to low-concentration alcohol, and finally distilled water. Then immerse the sections in hematoxylin solution for several minutes. Separate the sections with acid and ammonia solution for a few seconds each. Rinse with running water for 1 hour, then briefly in distilled water. Dehydrate in 70% and 90% alcohol for 10 minutes each. Stain with alcohol-eosin staining solution for 2-3 minutes.

[0384] 7. Dehydration, Clearing, and Sealing: After staining, the sections are dehydrated with pure alcohol and then cleared with xylene. Canada balsam is then applied to the cleared sections, and a coverslip is used to seal them. Once the balsam has slightly dried, a label is attached, and the specimen is ready for use.

[0385] Filming under a microscope, such as Figure 10 As shown, the results indicate that 76F-MDA5-R2P1-E10 can stimulate lung inflammation in mice.

[0386] This disclosure is not intended to limit itself to the specific embodiments disclosed herein, but is provided to illustrate aspects of this disclosure, for example. Various modifications to the compositions and methods will become apparent from the description and teaching herein. Such changes may be practiced without departing from the true scope and spirit of this disclosure, and are intended to fall within the scope of this disclosure.

Claims

1. An isolated anti-MDA5 antibody or its antigen-binding fragment, comprising a light chain variable region and a heavy chain variable region, wherein, The anti-MDA5 antibody or its antigen-binding fragment is any one of the following: (a) The light chain variable region comprises: CDR1-3 sequences as shown in SEQ ID NO: 3-5, and the heavy chain variable region comprises: CDR1-3 sequences as shown in SEQ ID NO: 6-8; (b) The light chain variable region comprises: CDR1, 3, 2 sequences as shown in SEQ ID NO: 41, 43 and KVS, and the heavy chain variable region comprises: CDR1~3 sequences as shown in SEQ ID NO: 44~46; The light chain variable region and the heavy chain variable region are coded according to the IMGT analysis method.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment comprises the following sequence: The variable region of the heavy chain is shown in SEQ ID NO: 22, and the variable region of the light chain is shown in SEQ ID NO: 21; or, the variable region of the heavy chain is shown in SEQ ID NO: 59, and the variable region of the light chain is shown in SEQ ID NO:

42.

3. The antibody or its antigen-binding fragment according to claim 2, wherein, The antibody or its antigen-binding fragment comprises the following sequence: The heavy chain is shown in SEQ ID NO: 18, and the light chain is shown in SEQ ID NO: 20; or, the heavy chain is shown in SEQ ID NO: 56, and the light chain is shown in SEQ ID NO:

58.

4. A polynucleotide, wherein, The polynucleotide encodes the antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

5. A carrier, wherein, The vector comprises the polynucleotide according to claim 4.

6. An isolated host cell, wherein, The host cell comprises the vector as described in claim 5.

7. A method for preparing host cells stably expressing a target protein in vitro, wherein, The method includes the step of transforming an initial host cell using the vector of claim 5.

8. A method for preparing a target protein in vitro, the method comprising preparing the target protein using the host cell of claim 6 or by the method of claim 7.

9. The antibody or its binding fragment prepared by the method according to claim 8.

10. A method for detecting anti-MDA5 antibodies for non-disease diagnostic and therapeutic purposes, wherein, The method includes the step of detecting the sample to be tested using the antibody or antigen-binding fragment thereof as described in any one of claims 1-3 or claim 9.

11. The method according to claim 10, wherein, The method includes the step of quantifying the anti-MDA5 antibody in the sample to be tested.

12. A reagent kit, wherein, The kit contains an antibody or antigen-binding fragment thereof according to any one of claims 1-3 or claim 9.

13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3 or claim 9 in the preparation of a reagent or kit for detecting anti-MDA5 antibody.

Images