CN114835813A

CN114835813A - Human antibody of dermatomyositis specific antigen peptide, preparation method and application

Info

Publication number: CN114835813A
Application number: CN202210259743.5A
Authority: CN
Inventors: 王雅楠; 杨衡; 王迁; 段利华; 曾小峰
Original assignee: Suzhou Institute Of Systems Medicine; Suzhou Fangke Biotechnology Co ltd; Peking Union Medical College Hospital Chinese Academy of Medical Sciences; Jiangxi Provincial Peoples Hospital
Current assignee: Suzhou Institute Of Systems Medicine; Suzhou Fangke Biotechnology Co ltd; Peking Union Medical College Hospital Chinese Academy of Medical Sciences; Jiangxi Provincial Peoples Hospital
Priority date: 2022-03-16
Filing date: 2022-03-16
Publication date: 2022-08-02
Also published as: CN116284371A

Abstract

The disclosure relates to a human antibody of dermatomyositis specific antigen peptide, a preparation method and application. In particular, the disclosure relates to a monoclonal antibody, the antibody specifically binds to MDA5 antigen protein, a preparation method of the monoclonal antibody and application of the monoclonal antibody in preparing a medicament for diagnosing, preventing or treating inflammatory myopathy or complications thereof. The antibody or antigen binding fragment of anti-MDA 5 screened by the method specifically binds to MDA5 antigen, can be used as a reference standard for qualitatively detecting MDA5 positivity, realizes qualitative or quantitative detection of anti-MDA 5 autoantibody level in patients with inflammatory myopathy or complications thereof, and has important significance for clinical diagnosis, disease monitoring and treatment of patients with DM, CAMD, DM-ILD, CADM-ILD and the like.

Description

Human antibody of dermatomyositis specific antigen peptide, preparation method and application

Technical Field

The disclosure belongs to the field of biomedicine, and relates to a human antibody of a specific antigenic peptide of dermatomyositis, a preparation method and application thereof. In particular, the disclosure relates to a monoclonal antibody, the antibody specifically binds to MDA5 antigen protein, a preparation method of the monoclonal antibody and application of the monoclonal antibody in preparing a medicament for diagnosing, preventing or treating inflammatory myopathy or complications thereof.

Background

Inflammatory Myopathy (IM) is a heterogeneous group of diseases characterized primarily by infiltration of skeletal myositis cells and necrosis of muscle fibers. Dermatomyositis (DM) is the most common subtype of inflammatory myopathy, primarily manifested by varying degrees of skin, muscle and lung involvement, with a incidence of (1-6)/10 million for adult DM. A common complication of DM is pulmonary interstitial diseases (ILD), with an incidence of 23.1% -65%. A clinically observed class of DM without significant muscle involvement is called clinical myomyopathic dermatomyositis (CADM). When CADM patients are combined with Acute Interstitial Pneumonia (AIP), the medicine has the characteristics of acute onset, rapid progression and high fatality rate, and the mortality rate of the patients in 6 months reaches 50%.

Myositis-specific antibodies (MSAs) have been increasingly detected in the serum of IM patients since the 70's of the 20 th century. The primary antibodies to DM include anti-Mi-2 antibodies, anti-transcription mediator 1-gamma antibodies, anti-MDA 5 antibodies, and anti-matrix protein 2 antibodies. MDA5 is an intracellular sensor of viral RNA (including coronaviruses) and is a protein encoded by melanoma differentiation associated gene 5 (MDA 5) that triggers innate immune responses. The CADM-140 antibody is screened from serum antibodies of patients with connective tissue disease and idiopathic ILD in the early 21 st century, and the antigen recognized by the antibody is MDA5 protein. anti-MDA 5 antibody was present in the sera of 35% DM patients and 75% CADM patients, and the incidence of antibody-positive patients with rapidly progressive ILD was significantly higher than that of antibody-negative patients (50%: 6%, P ═ 0.008).

Studies have shown that anti-MDA 5 antibody levels are closely related to the severity of skin ulcers, ILD disease severity and treatment, prognosis in patients with DM, CADM. In patients with anti-MDA 5 antibody levels ≧ 500U/mL, the ulcer was multiple, deep, whereas ulcers were more superficial and single in patients with antibody levels < 500U/mL. The incidence of AIP was higher in DM and CADM patients positive for anti-MDA 5 antibody (4% -33%) relative to anti-MDA 5 antibody negative patients (AIP incidence of about 20%), suggesting that anti-MDA 5 antibody is a serological marker of AIP. The incidence of ILD in patients with DM and CADM positive for the anti-MDA 5 antibody (42% -100%) was significantly higher than in patients with anti-MDA 5 antibody negative (10% -30%), and most patients had an acute or subacute clinical pattern of disease with ILD combined, dying from ILD and its complications within a short period of time (within 6 months). In addition, patients with high titer anti-MDA 5 antibody CADM-ILD had a poor prognosis, and after immunotherapy, the anti-MDA 5 antibody titer was significantly reduced or even disappeared. Therefore, the quantitative detection of the anti-MDA 5 antibody is beneficial to the early judgment of ILD progress and has significance for monitoring disease activity and evaluating treatment effect.

The detection methods aiming at the anti-MDA 5 antibody in China are both qualitative immunoblotting and immunomembrane strip methods, the consistency of the detection results is poor, and the anti-MDA 5 antibody level in the serum of a patient cannot be quantitatively detected. Therefore, how to rapidly and accurately diagnose the diseases and quantitatively detect the anti-MDA 5 antibody level in the serum of patients is an important problem to be solved.

Humanized antibodies are the main direction of development of therapeutic antibodies, and the emergence of phage antibody library technology provides a good technical platform for the preparation of humanized antibodies and gradually becomes one of the main means for obtaining humanized antibodies at present. The phage display technology is established for the first time by Smith in 1985, and has been widely applied to establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, antigen epitope research, cell signal transduction research and the like after development and perfection for more than thirty years. The phage antibody library technology is to amplify the complete set of variable region genes of antibodies by using Polymerase Chain Reaction (PCR), express Fab segments or single-chain antibodies (ScFv) on the surface of phage by phage surface display technology, thereby screening and enriching specific antibodies. It has been proposed that almost all possibilities for recombinant human monoclonal antibodies specifically reactive with antigens can be screened from a single pot antibody library system. Thus, when using phage antibody technology, a variety of antibody fragments can be obtained that can be applied for in vivo diagnosis or therapy.

Disclosure of Invention

Problems to be solved by the invention

Based on the problems in the prior art, the human monoclonal antibody aiming at the anti-dermatomyositis specific antigen MDA5 is screened out by the method.

Means for solving the problems

(1) An isolated antibody or antigen-binding fragment thereof against MDA5 comprising a light chain variable region, wherein the light chain variable region comprises one or more of the sequences set forth in seq id no:

(a1) as shown in SEQ ID NO: 3;

(a2) and SEQ ID NO:3, and 1, 2 or 3 conservative mutations in the amino acid sequence;

(a3) as shown in SEQ ID NO: 4;

(a4) and SEQ ID NO: 4, and 1 or 2 conservative mutations in the amino acid sequence;

(a5) as shown in SEQ ID NO: 5;

(a6) and SEQ ID NO: 5, and 1, 2 or 3 conservative mutations in the amino acid sequence;

the light chain variable region is encoded according to the IMGT analysis method.

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

(b1) as shown in SEQ ID NO: 6;

(b2) and SEQ ID NO: 6, and 1, 2 or 3 conservative mutations exist in the amino acid sequence;

(b3) as shown in SEQ ID NO: 7;

(b4) and SEQ ID NO: 7, wherein 1, 2 or 3 conservative mutations exist in the amino acid sequence;

(b5) as shown in SEQ ID NO: 8;

(b6) and SEQ ID NO: 8, and 1, 2 or 3 conservative mutations of the amino acid sequence;

the heavy chain variable region is encoded according to the IMGT analysis method.

(3) The antibody or antigen-binding fragment thereof of (1) or (2), comprising a heavy chain variable region comprising LCDR1, LCDR2 and LCDR3 and a light chain variable region comprising HCDR1, HCDR2 and HCDR 3; and the number of the first and second electrodes,

the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, and the LCDR2 comprises the amino acid sequence set forth as SEQ ID NO: 4, the LCDR3 comprising the amino acid sequence set forth as SEQ ID NO: 5;

the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 6, and the HCDR2 comprises the amino acid sequence shown as SEQ ID NO: 7, said HCDR3 comprising the amino acid sequence set forth as SEQ ID NO: 8.

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

(i) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 21;

(ii) (ii) a sequence which is conservatively mutated compared to the sequence set out in (i);

preferably, the antibody or antigen-binding fragment thereof comprises one or more of the sequences shown below:

(iii) the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:18, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20;

(iv) (iv) a sequence with conservative mutations compared to the sequence shown in (iii).

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

(a) comprises the amino acid sequence shown as SEQ ID NO: 17. SEQ ID NO:19 or a combination thereof;

(b) comprises the amino acid sequence shown as SEQ ID NO: 17. SEQ ID NO:19 or a combination thereof;

(c) a reverse complement of a sequence that is capable of hybridizing to the nucleotide sequence set forth in any one of (a) - (b) under high stringency hybridization conditions or very high stringency hybridization conditions;

(d) a sequence having at least 90% sequence identity to a nucleotide sequence set forth in any one of (a) - (c).

(6) A vector, wherein said vector comprises the polynucleotide according to (5).

(7) An isolated host cell, wherein said host cell comprises the vector of (6).

(8) A method for producing a host cell stably expressing a target protein, wherein said method comprises the step of transforming a starting host cell with the vector of (6).

(9) A method for producing a protein of interest, which comprises producing the protein of interest using the host cell of (7) or by the method of (8).

(10) An antibody or binding fragment thereof produced according to the method of (9).

(11) A method for detecting an anti-MDA 5 antibody, wherein the method comprises the step of detecting a sample to be tested using the antibody or antigen-binding fragment thereof according to any one of (1) to (4) or (10);

optionally, the method comprises the step of quantifying the anti-BP 180 antibody in the sample to be tested.

(12) A kit, wherein the kit comprises an antibody or antigen-binding fragment thereof according to any one of (1) to (4) or according to (10).

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

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

(1) detecting an anti-MDA 5 antibody, or preparing a reagent or kit for detecting an anti-MDA 5 antibody;

(2) preparing a reagent or a kit for diagnosing inflammatory myopathy or complications thereof;

(3) preparing a reagent or kit for monitoring the progression of inflammatory myopathy or its complications;

(4) preparing a reagent or a kit for researching the pathogenic mechanism of the inflammatory myopathy or the complication thereof;

optionally, the inflammatory myopathy is selected from at least one of: dermatomyositis DM, sarcoidosis-free dermatomyositis CADM; optionally, the complication is selected from at least one of: pulmonary interstitial lesion ILD, acute interstitial pneumonia AIP.

(15) A method for preventing or treating inflammatory myopathy or a complication thereof, wherein the antibody or antigen-binding fragment thereof according to any one of (1) to (4) or (10), or the composition according to (13) is administered to a subject;

ADVANTAGEOUS EFFECTS OF INVENTION

The human monoclonal antibody of the anti-dermatomyositis specific antigen MDA5 is obtained through screening. The antibody has high activity and good stability, has stronger specificity of combining with MDA5, can be used as a reference standard for qualitatively detecting the positive of an anti-MDA 5 antibody by combining with MDA5, can also quantitatively detect the level of an anti-MDA 5 autoantibody in patients with inflammatory myopathy or complications thereof, provides effective basis for diagnosis, disease monitoring and prognosis evaluation of patients with DM, CAMD, DM combined ILD (also known as DM-ILD), CAMD combined ILD (also known as CADM-ILD) and the like, disease mechanism research and the like, and is used for clinical diagnosis and treatment of patients with IM and IM-ILD.

Drawings

FIG. 1 shows a PCR agarose gel electrophoresis image of a monoclonal bacterium of a VL phage library; wherein Lane M is DL2000, Lane 1-16 is 21000076F pATA-VK, Lane17-32 is 21000076F pATA-V lambda.

FIG. 2 shows PCR agarose gel electrophoresis of a monoclonal bacterium of KH phage library; wherein Lane M is DL2000, Lane 1-48 is 21000076F pATA-scFv-KH.

FIG. 3 shows a PCR agarose gel electrophoresis image of a monoclonal bacterium of a lambda H phage library; wherein Lane M is DL2000, Lane 1-48 is 21000076F pATA-scFv-Lambda H.

FIG. 4 shows the results of monoclonal sequencing analysis of phage display libraries; wherein, the left graph is the analysis result of the library sequence light chain, and the right graph is the analysis result of the library sequence heavy chain.

FIG. 5 shows an SDS-PAGE electrophoresis of recombinant protein MDA 5; the protein size is 130KDa, and the purity is more than 95%.

Figure 6 shows the ELISA results at different concentrations of antibody dilution.

FIG. 7 shows the results of Western blot analysis of the expression of 76F-MDA5-R2P1-G5 antibody in A549 cells and RAW cells.

Detailed Description

Definition of

In the claims and/or the description of the present disclosure, the words "a" or "an" or "the" may mean "one", but may also mean "one or more", "at least one", and "one or more than one".

As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Also, the terms "comprising," "having," "including," or "containing" are intended to be inclusive and mean that there may be additional, unrecited elements or method steps.

Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.

The term "inflammatory myopathy," also known as "inflammatory myopathy, IM," is a group of heterogeneous diseases characterized primarily by the infiltration of skeletal myositis cells and necrosis of muscle fibers. "inflammatory myopathy" and "IM" are used interchangeably.

The term "dermatomyositis" also known as dermtomyositis, DM, is a non-suppurative inflammatory disorder mainly involving striated muscles and mainly involving lymphocyte infiltration, with or without various skin lesions. The terms "dermatomyositis" and "DM" may be used interchangeably.

The term "sarcoidosis-type dermatomyositis," also known as "crinically amyopathic dermatomyositis, CADM," refers to the type of dermatomyositis with only, or predominantly, skin lesions. "sarcopenia" and "CADM" may be used interchangeably.

The term "pulmonary interstitial disease" also known as "interstitial lung diseases, ILD" refers to inflammatory diseases of the pulmonary interstitium caused by various reasons, and the pathological changes mainly affect the pulmonary interstitium, and also affect the alveolar epithelial cells and the pulmonary blood vessels. "pulmonary interstitial lesions" and "ILDs" may be used interchangeably.

The term "acute interstitial pneumonia" is also called "acute interstitial pulmonary disease (AIP"), and is a rapidly developing fulminant lung injury, which is an acute injury lesion of the lung. "acute interstitial pneumonia" and "AIP" may be used interchangeably.

The terms "individual", "patient" or "subject" as used in the context of the present invention include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

As used in this disclosure, the terms "MDA 5", "MDA 5 antigen", "MDA 5 protein" are used interchangeably. MDA5 has 2N-terminal pattern recognition receptors (CARDs) and an RNA helicase domain, the CARD domain is responsible for signaling Toll-like receptor transduction, and the helicase domain is responsible for recognizing viral RNA. Virus-infected cells, particularly fibroblasts, dendritic cells and macrophages, highly express MDA5, recognize virus-derived nucleic acid molecules within the cytoplasm, initiate the type I interferon pathway, and lead to the production of a range of inflammatory mediators.

As used in the present disclosure, the term "conservative mutation" refers to a mutation (e.g., substitution, insertion, and/or deletion of amino acids) that can normally maintain the function of a protein. Illustratively, a conservative mutation is a conservative substitution.

As used in this disclosure, "conservative substitutions" typically exchange one amino acid at one or more positions in a protein. Such substitutions may be conservative. Examples of the substitution regarded as conservative substitution include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His or Lys, substitution of Asn with Glu, Gln, Lys, His or Asp, substitution of Asp with Asn, Glu or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp or Arg, substitution of Glu with Gly, Asn, Gln, Lys or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg or Tyr, substitution of Ile with Leu, Met, Val or Phe, substitution of Leu with Ile, Met, Val or Phe, substitution of Lys with Asn, Glu, Gln, His or Arg, substitution of Met with Met, Leu, Val or Phe, substitution of Phe with Trp, Tyr, Met, Ile or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe, Tyr, His, Val or Phe, and substitution of Met with Phe or Phe. Furthermore, conservative mutations include naturally occurring mutations due to individual differences in the origin of the gene, differences in strain, species, and the like.

"sequence identity" and "percent identity" in the present disclosure refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions in the aligned polynucleotides or polypeptides containing the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotides or polypeptides containing different nucleotide or amino acid residues. Polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or deleted nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or both polynucleotides). Polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or deleting amino acids (i.e., amino acid insertions or amino acid deletions in one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

The term "phage display technology" in the present disclosure is a biological technology in which a DNA sequence of a foreign protein or polypeptide is inserted into an appropriate position of a structural gene of a coat protein of a bacteriophage, so that the foreign gene is expressed in accordance with the expression of the coat protein, and at the same time, the foreign protein is displayed on the surface of the bacteriophage in accordance with the reassembly of the bacteriophage.

The term "antibody" in the present disclosure is used herein in the broadest sense to refer to a protein comprising 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.

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

The term "single chain antibody" (scFv) in the present disclosure is an antibody in which the variable region of the heavy chain and the variable region of the light chain of an antibody are linked by a short peptide (also called linker) of a limited number of amino acids.

The term "Fab" fragment in this disclosure includes the heavy and light chain variable domains and also includes the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F (ab') ₂ Antibody fragments were originally produced as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments.

The term "complementarity determining region" or "CDR" is a region of an antibody variable domain that is mutated in sequence and forms structurally defined loops ("hypervariable loops") and/or contains antigen-contacting residues ("antigen-contacting points"). The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR 3. 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 one or combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et Al (1989) Nature 342: 877- & 883, Al-Lazikani et Al, "Standard constraints for the structural characterization of Immunological constructs", Journal of Molecular Biology, 273, 927- & 948(1997)), based on antibody sequence variations Kabat (Kabat et Al, Sequences of Proteins of Immunological constructs, 4 th edition, U.S. Department of Health and Human Services, National instruments of Health (1987)), AbM (fundamental of Molecular), activity compatibility, London, Molecular Biology (IMGT), and the bulk of the clustering of CDs (International patent publication).

In the technical schemes described in the present disclosure, unless otherwise specified, all antibody numbering schemes employed in the present disclosure for antibodies are IMGT numbering schemes.

In some embodiments, the disclosure relates to stringency of hybridization conditions for defining the degree of complementarity of two polynucleotides. Alternatively, the aforementioned polynucleotide may be selected from DNA. "stringency" as used in this disclosure refers to the conditions of temperature and ionic strength during hybridization and the presence or absence of certain organic solvents. The higher the stringency, the higher the degree of complementarity between the target nucleotide sequence and the labeled polynucleotide sequence. "stringent conditions" refer to temperature and ionic conditions under which only nucleotide sequences having a high frequency of complementary bases will hybridize. The term "hybridizes under high or very high stringency conditions" as used herein describes the conditions used for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in molecular μ Lar Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6. Specific hybridization conditions mentioned in this disclosure are as follows: 1) high stringency hybridization conditions: washing one or more times in 6X sodium chloride/sodium citrate (SSC) at about 45 ℃ and then 0.2X SSC, 0.1% SDS at 65 ℃; 2) very high stringency hybridization conditions: 0.5M sodium phosphate, 7% SDS at 65 ℃ and then washed one or more times with 0.2 XSSC, 1% SDS at 65 ℃.

Technical scheme

In the technical scheme of the disclosure, the meanings represented by the numbers of the nucleotide and amino acid sequence table in the specification are as follows:

SEQ ID NO:1 is the amino acid sequence of MDA5 antigen;

the amino acid sequence of SEQ ID NO:2 is a nucleotide sequence for coding MDA5 antigen;

SEQ ID NO:3 is the amino acid sequence of 76F-MDA5-R2P1-G5 antibody LCDR 1;

SEQ ID NO: 4 is the amino acid sequence of 76F-MDA5-R2P1-G5 antibody LCDR 2;

SEQ ID NO: 5 is the amino acid sequence of 76F-MDA5-R2P1-G5 antibody LCDR 3;

SEQ ID NO: 6 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody HCDR 1;

SEQ ID NO: 7 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody HCDR 2;

SEQ ID NO: 8 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody HCDR 3;

SEQ ID NO: 9 is the amino acid sequence of the VL FR1 antibody 76F-MDA5-R2P 1-G5;

SEQ ID NO: 10 is the amino acid sequence of the VL FR2 antibody 76F-MDA5-R2P 1-G5;

SEQ ID NO: 11 is the amino acid sequence of the VL FR3 antibody 76F-MDA5-R2P 1-G5;

SEQ ID NO: 12 is the amino acid sequence of the VL FR4 antibody 76F-MDA5-R2P 1-G5;

SEQ ID NO: 13 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody VH FR 1;

SEQ ID NO: 14 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody VH FR 2;

SEQ ID NO: 15 is a nucleotide sequence encoding 76F-MDA5-R2P1-G5 antibody VH FR 3;

SEQ ID NO: 16 is the amino acid sequence of the 76F-MDA5-R2P1-G5 antibody VH FR 4;

SEQ ID NO:17 is a nucleotide sequence encoding the heavy chain of the 76F-MDA5-R2P1-G5 antibody;

SEQ ID NO:18 is the amino acid sequence of the heavy chain of the 76F-MDA5-R2P1-G5 antibody;

SEQ ID NO:19 is a nucleotide sequence encoding the light chain of antibody 76F-MDA5-R2P 1-G5;

SEQ ID NO:20 is the amino acid sequence of the light chain of the 76F-MDA5-R2P1-G5 antibody;

SEQ ID NO: 21 is a nucleotide sequence encoding the VH of antibody 76F-MDA5-R2P 1-G5;

SEQ ID NO: 22 is the amino acid sequence of antibody VL of 76F-MDA5-R2P 1-G5;

SEQ ID NO: 23-40 is a primer sequence.

anti-MDA 5 antibody or antigen-binding fragment thereof

anti-MDA 5 antibodies were present in the sera of DM and CADM patients, and the incidence of acute-progressive ILD associated with anti-MDA 5 antibody positive patients was significantly higher than antibody negative patients. The MDA5 antibody positive dermatomyositis is a rare autoimmune disease with extremely high fatality rate, and the subtype takes MDA5 antibody as a serological marker. Due to the rapid rate of progression of pulmonary disorders, patients have a six-month survival rate of only about 50% and often die from respiratory failure due to ILD. At present, the pathogenesis of the MDA5 antibody-positive dermatomyositis is not clear in the medical community, and the mainstream treatment scheme adopting hormone and the traditional immunosuppressant is not ideal in curative effect, so that the survival rate of the patient suffering from the MDA5 antibody-positive dermatomyositis and the rapid-progressing interstitial pneumonia cannot be obviously improved. The classical hybridoma technology is a long-period and strong-continuity experimental technology system. The method has the problems that a long-time preparation process and incomplete epitope recognition require subsequent humanized modification, so that a high-affinity humanized monoclonal antibody is difficult to obtain, and the level of the antibody cannot be absolutely quantified. These treatment modalities are therefore lack of specificity, poor in efficacy and long in course of treatment.

The phage antibody library technology can be used for in vitro library construction and screening of B cell antibody libraries of human and other animals, and avoids the steps of immunization, cell fusion and the like, so that the experimental period is shortened, the stability is increased, and the screening of a fully human antibody sequence with higher affinity in a short time is realized. In the disclosure, PBMCs of a patient with positive MDA5 antibody are separated to construct a phage humanized antibody library, and a humanized anti-MDA 5 antigen monoclonal antibody is screened out. The antibody can be specifically combined with MDA5, so that the level of anti-MDA 5 autoantibody in DM patients can be quantitatively detected, and the disease activity can be monitored and used for clinical diagnosis of DM patients. The research provides theoretical and experimental basis for specific treatment of DM, and also provides a new research direction for treating autoimmune diseases taking autoantibodies as main pathogenic mechanisms.

In some embodiments, the present disclosure produces MDA5 protein. In some embodiments, the step of preparing MDA5 protein comprises:

(1) recombining the gene of the artificially synthesized MDA5 structural domain into an expression vector plasmid pFastBac1 to obtain an MDA5-pFastBac1 expression vector;

(2) and transfecting the MDA5-pFastBac1 expression vector into a DH10Bac competence for culture, and purifying to obtain MDA5 antigen protein.

In some embodiments, the disclosure provides methods of screening for anti-MDA 5 antibodies, comprising the steps of:

(1) constructing a phage display library by using PBMCs derived from anti-MDA 5 antibody positive patients;

(2) screening the phage display library by using the MDA5 antigen protein to obtain the anti-MDA 5 antibody or the antigen binding fragment thereof, which is specifically combined with the anti-MDA 5 antigen protein.

In some more specific embodiments, the method of screening for an anti-MDA 5 antibody comprises:

PBMCs of a patient positive for MDA5 antibody were isolated, RNA was extracted and quality checked. And reverse transcribing the RNA qualified for quality inspection into cDNA by using an RT-PCR technology, and amplifying all antibody VH and VL gene segments to form a VK library and a V lambda library. Plasmid vectors of a VK library and a Vlambda library are extracted by using a plasmid extraction kit, and VH gene segments amplified in vitro are inserted into the plasmid vectors of the VK library and the Vlambda library to form a KH library and a lambda H library.

The antibody gene combinatorial library is inserted into the immediate downstream of the leader series of the gene III (g3) of the phage-encoded membrane protein, and the polypeptide or protein expressed by the exogenous antibody gene can be displayed at the N-terminal of the phage coat protein pIII in the form of fusion protein by assisting the superinfection of the phage. Each phage particle encodes and presents a different antibody, which contains billions of individual clones. In these antibody libraries, the genes encoding those antibodies that bind to the antigen are subjected to affinity enrichment-mild elution-phage amplification in vitro on the antigen, and the above enrichment and screening process is repeated until a library of antibody phage with good specificity and strong affinity is obtained after several cycles, and positive clones are screened from the library of antibody phage. And (3) identifying the positive clone by an ELISA method, and finally screening the fully human antibody with good specificity and strong affinity. And (3) carrying out western blotting on the human monoclonal antibody obtained by panning, a human non-small cell lung cancer cell line A549 and a mouse abdominal cavity macrophage cell line RAW, and respectively taking Gapdh and Actin as internal references to verify the antibody effect.

The method comprises the steps of screening three rounds of antibody phage libraries, taking clonidine of an antigen group which is more than 3 times that of a control group as positive clones, and carrying out sequencing analysis on the monoclonals. The wrong antibody sequences and repeated antibody sequences are eliminated, and the antibody is combined with the antigen-antibody specific binding capacity reflected by an ELISA experiment, so that 1 high-affinity antibody is finally obtained, and is named as 76F-MDA5-R2P 1-G5. Through ELISA and western blot verification, the antibody has high activity, good stability and stronger specificity, can be used as a reference standard for qualitatively detecting MDA5 positive, and can also be used for quantitatively detecting the anti-MDA 5 autoantibody level of patients with IM and IM-ILD (such as DM, CADM, DM-ILD, CADM-ILD and the like) sarcoidosis-free dermatomyositis.

Examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

All reagents used in the examples were commercially available unless otherwise noted.

The first embodiment is as follows: construction method of human ScFv phage display library

Table 1 main reagents used in this example

1. Library construction

1.1 Assembly of the heavy chain variable region (VH) and the light chain variable region (VL)

TABLE 2 PCR reaction conditions and procedures

Wherein, the three steps of denaturation, annealing and extension (1) are repeated for 30 times

The primer sequence is as follows:

Forward(F):

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

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

5′L-VH 4/6：CCCAGATGGGTCCTGTCCCAGGTGCAG(SEQ ID NO:25)

5′L-VH 5/7：CAAGGAGTCTGTTCCGAGGTGCAG(SEQ ID NO:26)

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

5′L VK 3：CTCTTCCTCCTGCTACTCTGGCTCCCAG(SEQ ID NO:28)

5′L VK 4/5：ATTTCTCTGTTGCTCTGGATCTCTG(SEQ ID NO:29)

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

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

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

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

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

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

5′L Vλ8/9/10：GAGTGGATTCTCAGACTGTGGTG(SEQ ID NO:36)

Reverse(R):

3′CK：TGCTGTCCTTGCTGTCCTGCT(SEQ ID NO:37)

3' C λ: CACCAGTGTGGCCTTGTTGGCTTG (SEQ ID NO:38)1.2 construction of a light chain variable region phage display library

1.2.1 preparation of pATA-scFv-2 vector as library clone

1.2.2 digestion vectors and PCR products

TABLE 3 digestion of vector and PCR product reaction System

1.2.3 connection

TABLE 4 ligation reaction System

Incubating overnight at 16 ℃ and heating to inactivate at 65 ℃ for 10 min.

1.2.4 electrotransfer

1.2.4.1 preparation of TG1 competent cells.

1mL SOC medium (Sigma, S1797) was prewarmed at 1.2.4.237 ℃. The electroporation cuvette (0.1 cm gap) and microcentrifuge tube were placed on ice (one cuvette and one microcentrifuge tube for each conversion reaction).

1.2.4.3 the Electrokinetic cells were removed from the freezer at-80 ℃ and placed on ice until they were completely thawed (10-15 min). After thawing, mix gently. 50 μ L of cells were placed in a frozen microcentrifuge tube on ice.

1.2.4.4 carefully add 3. mu.L of the DNA mixture to the frozen electroporation cuvette without generating air bubbles. Flick the tube down quickly with your wrist, depositing cells at the bottom.

1.2.4.5 electroporation at 600 Ω, 10 μ F and 1.8 kV. Within 10 seconds of the pulse, 1mL of pre-warmed SOC medium was added to each tube immediately. Shaking at 250rpm for 1 hour at 37 ℃.

1.2.4.6 collect all the electrotransformation medium. 10 μ L of the culture was serially diluted into 90 μ L SOC medium and plated on LB/Amp/Glucose. Incubate overnight at 37 ℃. The total number of transformants was calculated by counting the number of colonies, multiplying by the culture volume, and dividing by the plating volume.

1.3 construction of VL-VH phage display libraries

1.3.1 digestion vectors and PCR products

TABLE 5 digestion reaction System

1.3.2 connection

TABLE 6 ligation reaction System

Incubating overnight at 16 ℃ and heating to inactivate at 65 ℃ for 10 min.

1.3.3 electrotransfer

1.3.3.1 TG1 competent cells.

1.3.3.237 deg.C prewarmed 4mL SOC Medium (Sigma, S1797). The electroporation cuvette (0.2 cm gap) and microcentrifuge tube were placed on ice (one cuvette and one microcentrifuge tube for each conversion reaction).

1.3.3.3 the Electrokinetic cells were removed from the freezer at-80 ℃ and placed on ice until they were completely thawed (10-15 min). After thawing, mix gently.

1.3.3.4 carefully add 6. mu.L of the DNA mixture to the frozen electroporation cuvette without creating air bubbles. Flick the tube down quickly with your wrist, depositing the cells at the bottom.

1.3.3.5 electroporation at 600 Ω, 10 μ F and 2.5 kV. Within 10 seconds of the pulse, 2mL of pre-warmed SOC media was added to each tube immediately. Shaking at 250rpm for 1 hour at 37 ℃.

1.3.3.6 collect all the electrotransformation medium. 10 μ L of the culture was serially diluted into 90 μ L SOC medium and plated on LB/Amp/Glucose. Incubate overnight at 37 ℃. The total number of transformants was calculated by counting the number of colonies, multiplying by the culture volume, and dividing by the plating volume.

1.4 library evaluation

1.4.1 colony PCR: and carrying out PCR by using the constructed library as a template.

TABLE 7PCR reaction conditions

Wherein, the three steps of denaturation, annealing and extension (1) are repeated for 30 times.

The primer sequence is as follows:

Forward(F):AGCGGATAACAATTTCACACAGGA(SEQ ID NO:39)

Forward(R)：GCCCCCTTATTAGCGTTTGCCATC(SEQ ID NO:40)

the results of agarose gel electrophoresis after PCR are shown in FIGS. 1-3.

1.4.2 sequencing: selecting positive clones, and sequencing by Wuhan Pongzhike biotechnology, Inc., with the sequencing and quality control results shown in FIG. 4.

1.5 expression of MDA5 protein

Artificially synthesizing an MDA5 gene sequence, and recombining an MDA5 gene into an expression vector plasmid pFastBac1 to obtain an MDA5-pFastBac1 expression vector; the cloning site EcoRI/XbaI.

The amino acid sequence of MDA5 (SEQ ID NO:1) is:

MGSHHHHHHHHHHSGMSNGYSTDENFRYLISCFRARVKMYIQVEPVLDYLTFLPAEVKEQIQRTVATSGNMQAVELLLSTLEKGVWHLGWTREFVEALRRTGSPLAARYMNPELTDLPSPSFENAHDEYLQLLNLLQPTLVDKLLVRDVLDKCMEEELLTIEDRNRIAAAENNGNESGVRELLKRIVQKENWFSAFLNVLRQTGNNELVQELTGSDCSESNAEIENLSQVDGPQVEEQLLSTTVQPNLEKEVWGMENNSSESSFADSSVVSESDTSLAEGSVSCLDESLGHNSNMGSDSGTMGSDSDEENVAARASPEPELQLRPYQMEVAQPALEGKNIIICLPTGSGKTRVAVYIAKDHLDKKKKASEPGKVIVLVNKVLLVEQLFRKEFQPFLKKWYRVIGLSGDTQLKISFPEVVKSCDIIISTAQILENSLLNLENGEDAGVQLSDFSLIIIDECHHTNKEAVYNNIMRHYLMQKLKNNRLKKENKPVIPLPQILGLTASPGVGGATKQAKAEEHILKLCANLDAFTIKTVKENLDQLKNQIQEPCKKFAIADATREDPFKEKLLEIMTRIQTYCQMSPMSDFGTQPYEQWAIQMEKKAAKEGNRKERVCAEHLRKYNEALQINDTIRMIDAYTHLETFYNEEKDKKFAVIEDDSDEGGDDEYCDGDEDEDDLKKPLKLDETDRFLMTLFFENNKMLKRLAENPEYENEKLTKLRNTIMEQYTRTEESARGIIFTKTRQSAYALSQWITENEKFAEVGVKAHHLIGAGHSSEFKPMTQNEQKEVISKFRTGKINLLIATTVAEEGLDIKECNIVIRYGLVTNEIAMVQARGRARADESTYVLVAHSGSGVIEHETVNDFREKMMYKAIHCVQNMKPEEYAHKILELQMQSIMEKKMKTKRNIAKHYKNNPSLITFLCKNCSVLACSGEDIHVIEKMHHVNMTPEFKELYIVRENKALQKKCADYQINGEIICKCGQAWGTMMVHKGLDLPCLKIRNFVVVFKNNSTKKQYKKWVELPITFPNLDYSECCLFSDED；

the gene sequence is shown in SEQ ID NO. 2.

Transfecting an MDA5-pFastBac1 expression vector into a DH10Bac competence for culture, collecting precipitates, and performing GST tag affinity chromatography to obtain MDA5 protein; the purified MDA5 also needs to be subjected to SDS-PAGE (polyacrylamide gel electrophoresis) to verify its purity, and the SDS-PAGE of purified MDA5 is shown in FIG. 5, which is greater than 95% pure.

Example two: preparation of monoclonal antibody specifically binding to MDA5

Table 8 main reagents used in this example

Reagent	Numbering	Manufacturer(s) of
			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

1. First wheel

1.1 biopanning

1.1.1 coating: the immune tubes were coated and incubated overnight at 4 ℃. Antigen group: 1mL MDA5 transfection solution (50. mu.g/mL), control: mu.L of transfection solution (0. mu.g/mL).

1.1.2 washing: discard the tube and wash three times with 5mL of 0.05% PBST.

1.1.3 blocking: 5mL of 5% skim milk (dissolved in PBST) was added to the tube and incubated at 37 ℃ for 2 hours.

1.1.4 washing: discard the tube and wash with 5mL of 0.05% PBST.

1.1.5 incubation: the phage library was diluted with 1% skim milk (PBST lysis) and 1mL was added to the tube and incubated at 32 ℃ for 2 hours.

1.1.6 washing: discard the tube, wash three times with 5mL 0.05% PBST and two times with PBS.

1.1.7 elution: phage bound to MDA5 were eluted with 1mL glycine-hydrochloric acid (pH 2.2) and neutralized to pH 7.0 with Tris-HCl.

1.2 determination of the titer of diluted phages

1.2.1 E.coli TG1 was cultured until OD600 became 0.4-0.6.

1.2.2 mix 10. mu.L of diluted eluted phage with 190. mu.L of E.coli TG 1.

The mixture was incubated at 1.2.337 ℃ for 15 minutes and then poured into 2 XYT-A (Amp 100. mu.g/mL) medium. The medium was cultured in inverted condition at 37 ℃ overnight.

1.3 phage library amplification

1.3.1 mu.L of E.coli TG1 was added to 800. mu.L of 2YT broth and mixed cultured at 37 ℃ until OD600 became 0.4-0.6.

1.3.2 TG1, which had been cultured to the logarithmic phase, was transferred to 10mL of 2YT-G medium (final concentration of 2% glucose) and cultured on a shaker at 37 ℃ until OD600 became 0.4-0.6.

1.3.3 adding the eluted product, incubating at 37 ℃ for 30 minutes, and shake-culturing at 37 ℃ for 30 minutes.

1.3.4 adding 30mL 2YT-AG culture solution (final concentration of 0.1% Amp, 2% glucose), 37 degrees C shaking culture for 1 hours.

1.3.5M 13KO7(M13KO7: TG1 ═ 20:1) was added, and the mixture was incubated at 37 ℃ for 30 minutes and then shake-cultured at 37 ℃ for 30 minutes.

1.3.6 bacterial fluid in 5000rpm centrifugal 5 minutes. Resuspend with 40mL 2YT-AK (final concentration Amp 100. mu.g/mL, Kan 100. mu.g/mL) and incubate overnight at 30 ℃ with a shaker.

1.3.78000 rpm for 10 minutes, the supernatant was removed, resuspended in 1mL PBS, centrifuged at 12000rpm for 5 minutes, and the supernatant was transferred to a new 1.5mL centrifuge tube.

1.4 phage library titer assay after amplification

The procedure is as in 1.2

2. Second to third wheels

2.1 biopanning

And (3) repeating the step 1 twice in a circulating way, wherein the eluted phage after the previous round of amplification is used for each input phage library.

TABLE 9 biopanning results

3. Polyclonal phage ELISA

3.1 coating: the plate was coated and incubated overnight at 4 ℃. Antigen group: 100 μ L/well MDA5 protein (4 μ g/mL), control: 100 μ L/well protein dilution (0 μ g/mL).

3.2 washing: the microplate was discarded and each well was washed three times with 300. mu.L of 0.05% PBST.

3.3, sealing: mu.L of 5% skim milk (dissolved in PBS) was added to each well and blocked at 37 ℃ for 2 hours.

3.4 phage incubation: mu.L of diluted amplified phage was added to each well and incubated at 32 ℃ for 2 hours as shown in Table 10.

3.5 washing: the same as step 3.2.

3.6 incubation with secondary antibody: mu.L of anti-M13-HRP antibody (1:9000) diluted with blocking solution was added to each well and incubated at 32 ℃ for 1 hour.

3.7 washing: the same as step 3.2.

3.8 color development: the reaction was stopped by adding 100. mu.L of TMB to each well, incubating at room temperature, and then adding 50. mu.L of 2M HCl to each well.

3.9 reading plate: values were read at 450nm-630nm using a microplate reader.

TABLE 10 results of polyclonal phage ELISA

4. Monoclonal phage ELISA (selection of second round elution product for monoclonal based on polyclonal results)

4.1 96 clones were selected from the petri dish and cultured at 37 ℃ at 250rpm until OD600nm was 0.4-0.6.

4.2M13KO7 infected cultures (MOI 20:1), incubated at 37 ℃ for 30 min and shake-cultured at 37 ℃ for 30 min. The bacterial solution was centrifuged and resuspended in an equal volume of 2 XYT-AK (final Amp concentration 100. mu.g/mL, Kan 100. mu.g/mL) and incubated overnight at 30 ℃.

4.3 the culture was centrifuged and the supernatant was used for ELISA.

4.4 coating: the plate was coated and incubated overnight at 4 ℃. Antigen group: 100 μ L/well MDA5 protein (4 μ g/mL), control: 100 μ L/well protein dilution (0 μ g/mL).

4.5 washing: the microplate was discarded and each well was washed three times with 300. mu.L of 0.05% PBST.

4.6 sealing: mu.L of 5% skim milk (dissolved in PBS) was added to each well and blocked at 37 ℃ for 2 hours.

4.7 phage incubation: mu.L of phage supernatant was added to each well and incubated at 32 ℃ for 2 hours.

4.8 washing: the same as step 4.5.

4.9 incubation with secondary antibody: mu.L of anti-M13-HRP antibody (1:9000) diluted with blocking solution was added to each well and incubated at 32 ℃ for 1 hour.

4.10 washing: the same as step 4.5.

4.11 color development: the reaction was stopped by adding 100. mu.L of TMB to each well, incubating at room temperature, and then adding 50. mu.L of 2M HCl to each well.

4.12 plate reading: values were read at 450nm-630nm using a microplate reader and high specificity clones were sequenced.

TABLE 11 results of antigen group monoclonal phage ELISA

TABLE 12 results of control monoclonal phage ELISA

	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

5. Positive clone verification ELISA

5.1 mu.L of positive clones were added to 2mL of 2YT-AG medium (final concentration 0.1% Amp, 2% glucose) and cultured until OD600 became 0.4-0.6.

5.2M13KO7 infected cultures (MOI 20:1), incubated at 37 ℃ for 30 min and shake-cultured at 37 ℃ for 30 min. The bacterial solution was centrifuged and resuspended in an equal volume of 2 XYT-AK (final Amp concentration 100. mu.g/mL, Kan 100. mu.g/mL) and incubated overnight at 30 ℃.

5.3 the culture was centrifuged and the supernatant was used for ELISA.

5.4 coating: the plate was coated and incubated overnight at 4 ℃. Antigen group: 100 μ L/well MDA5 protein (4 μ g/mL), control: 100 μ L/well protein dilution (0 μ g/mL).

5.5 washing: the microplate was discarded and each well was washed three times with 300. mu.L of 0.05% PBST.

5.6 sealing: mu.L of 5% skim milk (dissolved in PBS) was added to each well and blocked at 37 ℃ for 2 hours.

5.7 phage incubation: mu.L of phage supernatant was added to each well and incubated at 32 ℃ for 2 hours.

5.8 washing: the same as step 4.5.

5.9 incubation with secondary antibody: mu.L of anti-M13-HRP antibody (1:9000) diluted with blocking solution was added to each well and incubated at 32 ℃ for 1 hour.

5.10 washing: the same as step 4.5.

5.11 color development: the reaction was stopped by adding 100. mu.L of TMB to each well, incubating at room temperature, and then adding 50. mu.L of 2M HCl to each well.

5.12 plate reading: values were read at 450nm-630nm using a microplate reader and high specificity clones were sequenced.

TABLE 13 results of positive monoclonal phage ELISA

The positive clone was sequenced and the sequence of the high specificity antibody obtained was as follows:

TABLE 1476F-MDA 5-R2P1-G5 antibody sequences

Sequencing of antibody sequences

Screening the obtained phage positive clones, and performing full sequence sequencing to obtain corresponding antibody heavy chain light chains, wherein the full sequence is as follows:

the base sequence of the heavy chain of the 76F-MDA5-R2P1-G5 antibody (SEQ ID NO:17) is:

GAATTCGCCGCCACCATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCTAGATGGGTGCTGAGCCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGACATCGGCGGTATGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGTGCTAGCACCAAGGGACCTTCTGTGTTCCCTCTGGCTCCTTCTTCTAAGTCCACTTCCGGTGGTACAGCAGCTCTGGGTTGTCTGGTGAAGGATTACTTCCCAGAACCAGTGACTGTGTCCTGGAACTCCGGAGCTCTGACTTCTGGAGTGCATACTTTCCCAGCAGTGCTGCAATCTAGCGGACTGTACTCTCTGTCTTCCGTGGTGACTGTGCCTTCTTCTTCCCTGGGGACTCAAACTTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCAAAGAGCTGCGATAAGACCCACACCTGTCCACCTTGTCCAGCTCCAGAACTGCTGGGTGGGCCTTCTGTGTTTCTGTTCCCACCTAAGCCAAAGGATACCCTGATGATCTCTAGGACCCCAGAAGTGACCTGTGTGGTCGTCGATGTGTCTCATGAAGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGGGTGGAAGTGCATAACGCAAAGACCAAGCCCAGGGAAGAGCAATACAACTCCACCTACAGGGTGGTCTCCGTCCTGACAGTCCTGCATCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAATAAAGCCCTGCCTGCCCCTATCGAGAAAACCATTAGCAAAGCCAAAGGCCAGCCCAGGGAGCCCCAGGTCTATACACTGCCCCCCAGCAGGGAGGAGATGACAAAAAATCAGGTCAGCCTGACATGCCTGGTCAAAGGCTTTTATCCCAGCGACATTGCCGTCGAGTGGGAGTCCAATGGCCAGCCCGAGAATAATTATAAAACAACACCCCCCGTCCTGGACAGCGACGGCAGCTTTTTTCTGTATAGCAAACTGACAGTCGATAAAAGCAGGTGGCAGCAGGGCAATGTCTTTTCCTGCAGCGTCATGCACGAGGCCCTGCACAATCACTATACTCAGAAAAGCCTGAGCCTGTCCCCCGGGAAATGAGCGGCCGC；

the heavy chain amino acid sequence of the 76F-MDA5-R2P1-G5 antibody (SEQ ID NO:18) is:

MKHLWFFLLLVAAPRWVLSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHRRYAFDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；

the light chain base sequence (SEQ ID NO:19) of the 76F-MDA5-R2P1-G5 antibody is:

GAATTCGCCGCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCTCTGCTGCTGTGGATCTCCGGCGCCTACGGCCAGGCTGTGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTATTGTAGCTCATATGTAGTCAGCTACACTTGGGTATTCGGCGGAGGCACCAAGGTGACCGTCCTCCGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC；

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

MVLQTQVFISLLLWISGAYGQAVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYVVSYTWVFGGGTKVTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

example three: ELISA detection of OD value of antibody under different dilution concentration conditions

ELISA experiment steps of enzyme-linked immunosorbent assay:

1. coating: 100 μ L/well MDA5 protein (4 μ g/mL) was coated on the plate and incubated overnight at 4 ℃.

2. Washing: the plate was discarded and each well was washed three times with 300. mu.L of 0.05% PBST.

3. And (3) sealing: mu.L of 5% skim milk (dissolved in PBS) was added to each well and blocked at 37 ℃ for 2 hours.

4. Positive antibody incubation: the 76F-MDA5-R2P1-H2 antibody was diluted in a gradient, and 100. mu.L of the diluted antibody solution was added to each well, followed by incubation at 37 ℃ for 1 hour.

5. Washing: the same as step 4.5.

6. And (3) secondary antibody incubation: goat Anti-Human IgG (H + L) antibody (Jackson, code: 109-.

7. Washing: the same as step 4.5.

8. Color development: the reaction was stopped by adding 100. mu.L of TMB to each well, incubating for 10min at 37 ℃ and then adding 50. mu.L of 2M HCl to each well.

9. Reading a plate: the value is read at 450nm-630nm by using an enzyme reader, and as shown in FIG. 6, the result shows that 76F-MDA5-R2P1-G5 has stronger specific binding capacity with MDA 5.

Example four: western blot verification of 76F-MDA5-R2P1-G5 antibody

Western blot experiment procedure:

1. cell collection: the cells were cultured to logarithmic phase, the cell pellet was collected, a suitable amount of lysis buffer was added to lyse the cells (phosphatase and protease inhibitor: RIPA lysis buffer: 1: 100), and incubated on ice for 30 minutes.

2. Protein extraction: the lysed cells were centrifuged at 12000rpm for 15 minutes at 4 ℃ and the supernatant was retained. To the supernatant was added a lysate volume of 1/3 (4 Xprotein SDS loading buffer), and after boiling at 100 ℃ for 10 minutes, the sample was stored at 4 ℃.

3. Preparing a lower layer adhesive: mixing 4 mL7.5% lower layer glue solution, 4 mL7.5% lower layer glue buffer solution and 80 muL coagulant, pouring the rubber plate, pressing the rubber by 1-2mL of absolute ethyl alcohol, waiting for solidification, and recovering the absolute ethyl alcohol.

4. Preparing a top layer adhesive: and (3) continuously pouring the rubber plate by using 1mL of the upper layer rubber solution, 1mL of the upper layer rubber buffer solution and 20 mu L of the coagulant, and waiting for 30 minutes until the rubber plate is solidified.

5. Electrophoresis: adding 30 mu L of protein into each hole, adjusting the voltage to 80V after loading, starting electrophoresis, adjusting the voltage to 120V after marker is leveled up, and stopping electrophoresis when the marker runs to the tail end of the gel plate.

6. Film transferring: 3 layers of filter paper are placed on the sponge, the glue is carefully placed on the filter paper, and then the membrane with the same size as the glue is placed above the filter paper to exhaust air bubbles. And (3) superposing 2 layers of filter paper and a layer of sponge, clamping the superposed sandwich, putting the sandwich into an electrophoresis tank, adding a transfer membrane solution, putting the electrophoresis tank into a foam box, and adding ice around the tank. Wet-rotating for 300A and 120 min.

7. And (3) sealing: 5% skim milk (TPBS dissolved) was blocked for 1 hour.

8. Primary antibody incubation: the 76F-MDA5-R2P1-G5 antibody was diluted 1:100 in antibody dilution and incubated overnight at 4 ℃.

9. Washing the membrane: 1XTBST washes were performed three times, each for 10 minutes.

10. And (3) secondary antibody incubation: goat Anti-Human IgG (H + L) antibody (Jackson, code: 109-.

11. Washing the membrane: the same as step 9.

12. And (4) luminescence detection imaging. As shown in fig. 7. The results showed that 76F-MDA5-R2P1-G5 was expressed in both A549 and RAW cells.

The present disclosure is not intended to be limited in scope by the specifically disclosed embodiments, which are provided, for example, to illustrate aspects of the present disclosure. Various modifications to the compositions and methods will be apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure, and are intended to fall within the scope of the disclosure.

SEQUENCE LISTING

<110> Suzhou Fagaku Biotechnology Ltd

Suzhou Institute of Systems Medicine

PEKING UNION MEDICAL College HOSPITAL CHINESE ACADEMY OF MEDICAL SCIENCES

JIANGXI PROVINCIAL PEOPLE'S Hospital

Human antibody of <120> dermatomyositis specific antigen peptide, preparation method and application

<130> 6A59-2118284IP

<160> 40

<170> PatentIn version 3.5

<210> 1

<211> 1040

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of MDA5

<400> 1

Met Gly Ser His His His His His His His His His His Ser Gly Met

1 5 10 15

Ser Asn Gly Tyr Ser Thr Asp Glu Asn Phe Arg Tyr Leu Ile Ser Cys

20 25 30

Phe Arg Ala Arg Val Lys Met Tyr Ile Gln Val Glu Pro Val Leu Asp

35 40 45

Tyr Leu Thr Phe Leu Pro Ala Glu Val Lys Glu Gln Ile Gln Arg Thr

50 55 60

Val Ala Thr Ser Gly Asn Met Gln Ala Val Glu Leu Leu Leu Ser Thr

65 70 75 80

Leu Glu Lys Gly Val Trp His Leu Gly Trp Thr Arg Glu Phe Val Glu

85 90 95

Ala Leu Arg Arg Thr Gly Ser Pro Leu Ala Ala Arg Tyr Met Asn Pro

100 105 110

Glu Leu Thr Asp Leu Pro Ser Pro Ser Phe Glu Asn Ala His Asp Glu

115 120 125

Tyr Leu Gln Leu Leu Asn Leu Leu Gln Pro Thr Leu Val Asp Lys Leu

130 135 140

Leu Val Arg Asp Val Leu Asp Lys Cys Met Glu Glu Glu Leu Leu Thr

145 150 155 160

Ile Glu Asp Arg Asn Arg Ile Ala Ala Ala Glu Asn Asn Gly Asn Glu

165 170 175

Ser Gly Val Arg Glu Leu Leu Lys Arg Ile Val Gln Lys Glu Asn Trp

180 185 190

Phe Ser Ala Phe Leu Asn Val Leu Arg Gln Thr Gly Asn Asn Glu Leu

195 200 205

Val Gln Glu Leu Thr Gly Ser Asp Cys Ser Glu Ser Asn Ala Glu Ile

210 215 220

Glu Asn Leu Ser Gln Val Asp Gly Pro Gln Val Glu Glu Gln Leu Leu

225 230 235 240

Ser Thr Thr Val Gln Pro Asn Leu Glu Lys Glu Val Trp Gly Met Glu

245 250 255

Asn Asn Ser Ser Glu Ser Ser Phe Ala Asp Ser Ser Val Val Ser Glu

260 265 270

Ser Asp Thr Ser Leu Ala Glu Gly Ser Val Ser Cys Leu Asp Glu Ser

275 280 285

Leu Gly His Asn Ser Asn Met Gly Ser Asp Ser Gly Thr Met Gly Ser

290 295 300

Asp Ser Asp Glu Glu Asn Val Ala Ala Arg Ala Ser Pro Glu Pro Glu

305 310 315 320

Leu Gln Leu Arg Pro Tyr Gln Met Glu Val Ala Gln Pro Ala Leu Glu

325 330 335

Gly Lys Asn Ile Ile Ile Cys Leu Pro Thr Gly Ser Gly Lys Thr Arg

340 345 350

Val Ala Val Tyr Ile Ala Lys Asp His Leu Asp Lys Lys Lys Lys Ala

355 360 365

Ser Glu Pro Gly Lys Val Ile Val Leu Val Asn Lys Val Leu Leu Val

370 375 380

Glu Gln Leu Phe Arg Lys Glu Phe Gln Pro Phe Leu Lys Lys Trp Tyr

385 390 395 400

Arg Val Ile Gly Leu Ser Gly Asp Thr Gln Leu Lys Ile Ser Phe Pro

405 410 415

Glu Val Val Lys Ser Cys Asp Ile Ile Ile Ser Thr Ala Gln Ile Leu

420 425 430

Glu Asn Ser Leu Leu Asn Leu Glu Asn Gly Glu Asp Ala Gly Val Gln

435 440 445

Leu Ser Asp Phe Ser Leu Ile Ile Ile Asp Glu Cys His His Thr Asn

450 455 460

Lys Glu Ala Val Tyr Asn Asn Ile Met Arg His Tyr Leu Met Gln Lys

465 470 475 480

Leu Lys Asn Asn Arg Leu Lys Lys Glu Asn Lys Pro Val Ile Pro Leu

485 490 495

Pro Gln Ile Leu Gly Leu Thr Ala Ser Pro Gly Val Gly Gly Ala Thr

500 505 510

Lys Gln Ala Lys Ala Glu Glu His Ile Leu Lys Leu Cys Ala Asn Leu

515 520 525

Asp Ala Phe Thr Ile Lys Thr Val Lys Glu Asn Leu Asp Gln Leu Lys

530 535 540

Asn Gln Ile Gln Glu Pro Cys Lys Lys Phe Ala Ile Ala Asp Ala Thr

545 550 555 560

Arg Glu Asp Pro Phe Lys Glu Lys Leu Leu Glu Ile Met Thr Arg Ile

565 570 575

Gln Thr Tyr Cys Gln Met Ser Pro Met Ser Asp Phe Gly Thr Gln Pro

580 585 590

Tyr Glu Gln Trp Ala Ile Gln Met Glu Lys Lys Ala Ala Lys Glu Gly

595 600 605

Asn Arg Lys Glu Arg Val Cys Ala Glu His Leu Arg Lys Tyr Asn Glu

610 615 620

Ala Leu Gln Ile Asn Asp Thr Ile Arg Met Ile Asp Ala Tyr Thr His

625 630 635 640

Leu Glu Thr Phe Tyr Asn Glu Glu Lys Asp Lys Lys Phe Ala Val Ile

645 650 655

Glu Asp Asp Ser Asp Glu Gly Gly Asp Asp Glu Tyr Cys Asp Gly Asp

660 665 670

Glu Asp Glu Asp Asp Leu Lys Lys Pro Leu Lys Leu Asp Glu Thr Asp

675 680 685

Arg Phe Leu Met Thr Leu Phe Phe Glu Asn Asn Lys Met Leu Lys Arg

690 695 700

Leu Ala Glu Asn Pro Glu Tyr Glu Asn Glu Lys Leu Thr Lys Leu Arg

705 710 715 720

Asn Thr Ile Met Glu Gln Tyr Thr Arg Thr Glu Glu Ser Ala Arg Gly

725 730 735

Ile Ile Phe Thr Lys Thr Arg Gln Ser Ala Tyr Ala Leu Ser Gln Trp

740 745 750

Ile Thr Glu Asn Glu Lys Phe Ala Glu Val Gly Val Lys Ala His His

755 760 765

Leu Ile Gly Ala Gly His Ser Ser Glu Phe Lys Pro Met Thr Gln Asn

770 775 780

Glu Gln Lys Glu Val Ile Ser Lys Phe Arg Thr Gly Lys Ile Asn Leu

785 790 795 800

Leu Ile Ala Thr Thr Val Ala Glu Glu Gly Leu Asp Ile Lys Glu Cys

805 810 815

Asn Ile Val Ile Arg Tyr Gly Leu Val Thr Asn Glu Ile Ala Met Val

820 825 830

Gln Ala Arg Gly Arg Ala Arg Ala Asp Glu Ser Thr Tyr Val Leu Val

835 840 845

Ala His Ser Gly Ser Gly Val Ile Glu His Glu Thr Val Asn Asp Phe

850 855 860

Arg Glu Lys Met Met Tyr Lys Ala Ile His Cys Val Gln Asn Met Lys

865 870 875 880

Pro Glu Glu Tyr Ala His Lys Ile Leu Glu Leu Gln Met Gln Ser Ile

885 890 895

Met Glu Lys Lys Met Lys Thr Lys Arg Asn Ile Ala Lys His Tyr Lys

900 905 910

Asn Asn Pro Ser Leu Ile Thr Phe Leu Cys Lys Asn Cys Ser Val Leu

915 920 925

Ala Cys Ser Gly Glu Asp Ile His Val Ile Glu Lys Met His His Val

930 935 940

Asn Met Thr Pro Glu Phe Lys Glu Leu Tyr Ile Val Arg Glu Asn Lys

945 950 955 960

Ala Leu Gln Lys Lys Cys Ala Asp Tyr Gln Ile Asn Gly Glu Ile Ile

965 970 975

Cys Lys Cys Gly Gln Ala Trp Gly Thr Met Met Val His Lys Gly Leu

980 985 990

Asp Leu Pro Cys Leu Lys Ile Arg Asn Phe Val Val Val Phe Lys Asn

995 1000 1005

Asn Ser Thr Lys Lys Gln Tyr Lys Lys Trp Val Glu Leu Pro Ile

1010 1015 1020

Thr Phe Pro Asn Leu Asp Tyr Ser Glu Cys Cys Leu Phe Ser Asp

1025 1030 1035

Glu Asp

1040

<210> 2

<211> 3138

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence of MDA5

<400> 2

gaattcacca tgggcagcca ccaccaccac catcaccacc accaccactc cggcatgagc 60

aacggctaca gcactgacga gaacttccgc tacctgatca gctgcttccg cgcccgcgtc 120

aagatgtaca tccaggtcga acctgtcctg gactacctga ccttcctgcc tgccgaggtg 180

aaggaacaga tccagcgtac cgtcgctact agcggtaaca tgcaggctgt ggagctgctg 240

ctgtccactc tggagaaggg tgtctggcac ctgggctgga cccgcgaatt tgtcgaagct 300

ctgcgccgta ccggcagccc tctggctgct aggtacatga accctgaact gaccgacctg 360

cccagcccct ccttcgaaaa cgctcacgac gaatacctgc agctgctgaa cctgctgcag 420

cccaccctgg tggacaagct gctggtgcgt gacgtcctgg acaagtgcat ggaagaggag 480

ctgctgacca tcgaggaccg taaccgcatc gccgctgccg aaaacaacgg taacgaaagc 540

ggtgtccgtg aactgctgaa gcgtatcgtg cagaaggaga actggttcag cgctttcctg 600

aacgtgctgc gccagactgg taacaacgag ctggtgcagg aactgaccgg ttccgactgc 660

tccgagagca acgctgagat cgagaacctg tcccaggtgg acggtcctca ggtggaggag 720

cagctgctgt ccaccactgt gcagcctaac ctggagaagg aagtctgggg catggagaac 780

aactcctccg agtcctcctt cgctgacagc agcgtggtgt ccgaatccga cacttccctg 840

gccgagggta gcgtgagctg cctggacgag agcctgggtc acaactccaa catgggctcc 900

gacagcggca ccatgggctc cgatagcgac gaagagaacg tcgctgcccg cgctagcccc 960

gaacccgaac tgcagctgcg cccctaccag atggaagtgg ctcagcctgc cctggaaggc 1020

aagaacatca tcatctgcct gcccactggt tccggcaaga cccgtgtcgc tgtgtacatc 1080

gctaaggacc acctggacaa gaagaagaag gcctccgaac ccggcaaggt cattgtgctg 1140

gtcaacaagg tcctgctggt cgaacagctg ttccgcaagg agttccagcc tttcctgaag 1200

aagtggtacc gcgtgatcgg tctgagcggt gacactcagc tgaagatcag cttccctgaa 1260

gtggtgaagt cctgcgacat catcatctcc accgctcaga tcctggaaaa ctccctgctg 1320

aacctcgaaa acggcgagga cgctggcgtc cagctgagcg acttctccct gatcatcatc 1380

gacgagtgcc accacaccaa caaggaagcc gtctacaaca acatcatgcg ccactacctg 1440

atgcagaagc tgaagaacaa ccgcctgaag aaggagaaca agcctgtcat ccctctgccc 1500

cagatcctgg gcctgaccgc ctcccctggc gtcggtggtg ctaccaagca ggctaaggcc 1560

gaggagcaca tcctgaagct gtgcgctaac ctggacgctt tcaccatcaa gaccgtgaag 1620

gagaacctgg accagctgaa gaaccagatc caggagccct gcaagaagtt cgccatcgct 1680

gacgctactc gcgaagaccc tttcaaggag aagctgctgg aaatcatgac tcgtatccag 1740

acttactgcc agatgtcccc catgtccgac ttcggtactc agccctacga gcagtgggct 1800

atccagatgg agaagaaggc cgccaaggag ggtaaccgca aggaacgcgt gtgcgctgaa 1860

cacctgcgta agtacaacga ggccctgcag atcaacgaca ctatccgcat gatcgacgct 1920

tacactcacc tggagacttt ctacaacgaa gagaaggaca agaagttcgc tgtgatcgag 1980

gacgactccg acgaaggtgg cgacgacgag tactgcgacg gtgacgaaga cgaagacgac 2040

ctgaagaagc ccctgaagct ggacgaaacc gaccgcttcc tgatgactct gttcttcgag 2100

aacaacaaga tgctgaagcg tctggccgag aaccctgaat acgaaaacga gaagctgact 2160

aagctgcgta acaccatcat ggaacagtac actcgcaccg aagaaagcgc tcgcggcatc 2220

atcttcacca agacccgtca gagcgcctac gccctgtccc agtggatcac cgaaaacgag 2280

aaattcgctg aggtgggcgt caaggcccac cacctgatcg gtgctggtca cagctccgag 2340

tttaaaccta tgacccagaa cgagcagaag gaagtcatca gcaagttccg tactggtaaa 2400

atcaacctgc tgatcgctac cactgtcgcc gaggagggcc tggacatcaa ggagtgcaac 2460

atcgtgatcc gttacggcct ggtgaccaac gaaatcgcca tggtgcaggc ccgcggtcgc 2520

gctagagccg atgaaagcac ctacgtgctg gtggctcact ccggtagcgg tgtgatcgag 2580

cacgagactg tgaacgactt ccgtgaaaag atgatgtaca aggccatcca ctgcgtgcag 2640

aacatgaagc ccgaagaata cgcccacaag atcctggaac tgcagatgca gtccatcatg 2700

gagaagaaaa tgaagaccaa gcgtaacatc gctaagcact acaagaacaa cccctccctg 2760

atcaccttcc tgtgcaagaa ctgcagcgtg ctggcttgct ccggcgagga catccacgtg 2820

atcgaaaaga tgcaccacgt caacatgact cccgagttta aagaactgta catcgtgcgt 2880

gagaacaagg ctctgcagaa gaagtgcgct gactaccaga tcaacggcga gatcatctgc 2940

aagtgcggtc aggcctgggg caccatgatg gtgcacaagg gtctggacct gccctgcctg 3000

aagatccgca acttcgtggt ggtcttcaag aacaactcca ctaagaagca gtacaagaag 3060

tgggtcgagc tgcctatcac cttccccaac ctggactact ccgagtgctg cctgttcagc 3120

gacgaggact aatctaga 3138

<210> 3

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VL CDR1

<400> 3

Ser Ser Asp Val Gly Gly Tyr Asn Tyr

1 5

<210> 4

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VL CDR2

<400> 4

Asp Val Ser

1

<210> 5

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VL CDR3

<400> 5

Ser Ser Tyr Val Val Ser Tyr Thr Trp Val

1 5 10

<210> 6

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VH CDR1

<400> 6

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 7

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VH CDR2

<400> 7

Ile Ser Tyr Asp Gly Ser Asn Lys

1 5

<210> 8

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VH CDR3

<400> 8

Ala Arg His Arg Arg Tyr Ala Phe Asp Ile

1 5 10

<210> 9

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VL FR1

<400> 9

Gln Ala Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr

20 25

<210> 10

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VL FR2

<400> 10

Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile

1 5 10 15

Tyr

<210> 11

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VL FR3

<400> 11

Lys Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly

1 5 10 15

Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala

20 25 30

Asp Tyr Tyr Cys

35

<210> 12

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VL FR4

<400> 12

Phe Gly Gly Gly Thr Lys Val Thr Val Leu

1 5 10

<210> 13

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VH FR1

<400> 13

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 14

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VH FR2

<400> 14

Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

1 5 10 15

Val

<210> 15

<211> 38

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VH FR3

<400> 15

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 16

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VH FR4

<400> 16

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<210> 17

<211> 1424

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence of heavy chain

<400> 17

gaattcgccg ccaccatgaa gcacctgtgg ttctttctgc tgctggtggc cgctcctaga 60

tgggtgctga gccaggtgca gctggtggag tctgggggag gcgtggtcca gcctgggagg 120

tccctgagac tctcctgtgc agcctctgga ttcaccttca gtagctatgc tatgcactgg 180

gtccgccagg ctccaggcaa ggggctagag tgggtggcag ttatatcata tgatggaagt 240

aataaatact acgcagactc cgtgaagggc cgattcacca tctccagaga caattccaag 300

aacacgctgt atctgcaaat gaacagcctg agagctgagg acacggctgt gtattactgt 360

gcgagacatc ggcggtatgc ttttgatatc tggggccaag ggaccacggt caccgtctcg 420

agtgctagca ccaagggacc ttctgtgttc cctctggctc cttcttctaa gtccacttcc 480

ggtggtacag cagctctggg ttgtctggtg aaggattact tcccagaacc agtgactgtg 540

tcctggaact ccggagctct gacttctgga gtgcatactt tcccagcagt gctgcaatct 600

agcggactgt actctctgtc ttccgtggtg actgtgcctt cttcttccct ggggactcaa 660

acttacatct gcaacgtgaa ccacaagccc tccaacacca aggtggacaa gaaggtggag 720

ccaaagagct gcgataagac ccacacctgt ccaccttgtc cagctccaga actgctgggt 780

gggccttctg tgtttctgtt cccacctaag ccaaaggata ccctgatgat ctctaggacc 840

ccagaagtga cctgtgtggt cgtcgatgtg tctcatgaag accctgaagt gaagttcaac 900

tggtacgtgg acggggtgga agtgcataac gcaaagacca agcccaggga agagcaatac 960

aactccacct acagggtggt ctccgtcctg acagtcctgc atcaggattg gctgaacggc 1020

aaggagtaca agtgcaaggt ctccaataaa gccctgcctg cccctatcga gaaaaccatt 1080

agcaaagcca aaggccagcc cagggagccc caggtctata cactgccccc cagcagggag 1140

gagatgacaa aaaatcaggt cagcctgaca tgcctggtca aaggctttta tcccagcgac 1200

attgccgtcg agtgggagtc caatggccag cccgagaata attataaaac aacacccccc 1260

gtcctggaca gcgacggcag cttttttctg tatagcaaac tgacagtcga taaaagcagg 1320

tggcagcagg gcaatgtctt ttcctgcagc gtcatgcacg aggccctgca caatcactat 1380

actcagaaaa gcctgagcct gtcccccggg aaatgagcgg ccgc 1424

<210> 18

<211> 466

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of heavy chain

<400> 18

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg His Arg Arg Tyr Ala Phe Asp Ile Trp Gly Gln

115 120 125

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

130 135 140

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

145 150 155 160

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

165 170 175

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

195 200 205

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

210 215 220

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

225 230 235 240

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Lys

465

<210> 19

<211> 737

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence of light chain

<400> 19

gaattcgccg ccaccatggt gctgcagacc caggtgttca tctctctgct gctgtggatc 60

tccggcgcct acggccaggc tgtgctgact cagcctgcct ccgtgtctgg gtctcctgga 120

cagtcgatca ccatctcctg cactggaacc agcagtgatg ttggtggtta taactatgtc 180

tcctggtacc aacagcaccc aggcaaagcc cccaaactca tgatttatga tgtcagtaag 240

cggccctcag gggtttctaa tcgcttctct ggctccaagt ctggcaacac ggcctccctg 300

acaatctctg ggctccaggc tgaggatgag gctgattatt attgtagctc atatgtagtc 360

agctacactt gggtattcgg cggaggcacc aaggtgaccg tcctccgtac ggtggctgca 420

ccttctgtgt tcatcttccc tccatctgat gagcagctga agtctggaac cgcatctgtc 480

gtctgtctgc tgaacaactt ttaccccagg gaggctaagg tccaatggaa ggtggacaac 540

gccctgcagt ctggtaatag ccaggaaagc gtgaccgaac aggattccaa ggactccacc 600

tactccctgt cctccacact gacactgagc aaagccgact atgaaaagca caaagtgtat 660

gcctgcgagg tcactcatca gggcctgtcc agccccgtga ctaaaagctt taataggggg 720

gagtgctgag cggccgc 737

<210> 20

<211> 237

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of light chain

<400> 20

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Gln Ala Val Leu Thr Gln Pro Ala Ser Val Ser Gly

20 25 30

Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp

35 40 45

Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys

50 55 60

Ala Pro Lys Leu Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val

65 70 75 80

Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr

85 90 95

Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser

100 105 110

Tyr Val Val Ser Tyr Thr Trp Val Phe Gly Gly Gly Thr Lys Val Thr

115 120 125

Val Leu Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

130 135 140

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

145 150 155 160

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

165 170 175

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

180 185 190

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

195 200 205

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

210 215 220

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 21

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VL

<400> 21

Gln Ala Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Val Val Ser

85 90 95

Tyr Thr Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 22

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of VH

<400> 22

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Arg Arg Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 23

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 23

acaggtgccc actcccaggt gcag 24

<210> 24

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 24

aaggtgtcca gtgtgargtg cag 23

<210> 25

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 25

cccagatggg tcctgtccca ggtgcag 27

<210> 26

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 26

caaggagtct gttccgaggt gcag 24

<210> 27

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 27

atgaggstcc cygctcagct gctgg 25

<210> 28

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 28

ctcttcctcc tgctactctg gctcccag 28

<210> 29

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 29

atttctctgt tgctctggat ctctg 25

<210> 30

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 30

ggtcctgggc ccagtctgtg ctg 23

<210> 31

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 31

ggtcctgggc ccagtctgcc ctg 23

<210> 32

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 32

gctctgtgac ctcctatgag ctg 23

<210> 33

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 33

ggtctctctc scagcytgtg ctg 23

<210> 34

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 34

gttcttgggc caattttatg ctg 23

<210> 35

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 35

ggtccaattc ycaggctgtg gtg 23

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 36

gagtggattc tcagactgtg gtg 23

<210> 37

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 37

tgctgtcctt gctgtcctgc t 21

<210> 38

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 38

caccagtgtg gccttgttgg cttg 24

<210> 39

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 39

agcggataac aatttcacac agga 24

<210> 40

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> primer sequence

<400> 40

gcccccttat tagcgtttgc catc 24

Claims

1. An isolated antibody or antigen-binding fragment thereof against MDA5 comprising a light chain variable region, wherein the light chain variable region comprises one or more of the sequences set forth in seq id no:

(a1) as shown in SEQ ID NO: 3;

(a2) and SEQ ID NO:3, wherein 1, 2 or 3 conservative mutations exist in the amino acid sequence;

(a3) as shown in SEQ ID NO: 4;

(a5) as shown in SEQ ID NO: 5;

2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region, wherein the heavy chain variable region comprises one or more of the sequences set forth in seq id no:

(b1) as shown in SEQ ID NO: 6;

(b3) as shown in SEQ ID NO: 7;

(b4) and SEQ ID NO: 7, wherein 1, 2 or 3 conservative mutations are present in the amino acid sequence;

(b5) as shown in SEQ ID NO: 8;

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region comprising LCDR1, LCDR2, and LCDR3 and a light chain variable region comprising HCDR1, HCDR2, and HCDR 3; and the number of the first and second electrodes,

the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, the LCDR2 comprising the amino acid sequence set forth as SEQ ID NO: 4, the LCDR3 comprising the amino acid sequence set forth as SEQ ID NO: 5;

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof comprises one or more of the following sequences:

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

(a) comprises a nucleotide sequence as set forth in SEQ ID NO: 17. the amino acid sequence of SEQ ID NO:19 or a combination thereof;

(b) comprises the amino acid sequence shown as SEQ ID NO: 17. the amino acid sequence of SEQ ID NO:19 or a combination thereof;

6. A vector, wherein said vector comprises the polynucleotide of claim 5.

7. An isolated host cell, wherein the host cell comprises the vector of claim 6.

8. A method for producing a host cell stably expressing a protein of interest, wherein the method comprises the step of transforming a starting host cell with the vector of claim 6.

9. A method of producing a protein of interest, the method comprising using the host cell of claim 7 or producing the protein of interest by the method of claim 8.

10. An antibody or binding fragment thereof prepared according to the method of claim 9.

11. A method of detecting an anti-MDA 5 antibody, wherein the method comprises the step of detecting a test sample using the antibody or antigen-binding fragment thereof of any one of claims 1-4 or claim 10;

optionally, the method comprises the step of quantifying the anti-BP 180 antibody in the test sample.

12. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-4 or claim 10.

13. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or claim 10.

14. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4 or claim 10, or the composition according to claim 13, in at least one of (1) - (4) below:

15. A method of preventing or treating inflammatory myopathy or a complication thereof, wherein the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or claim 10, or the composition according to claim 13 is administered to a subject;