Anti-MuSK protein antibody or related product and application thereof

Abstract

The invention discloses an anti-MuSK protein antibody or related products and application thereof, and relates to the field of antibodies. The anti-MuSK protein antibody provided by the invention shows extremely high affinity and specificity, can be accurately combined with a specific epitope of the MuSK protein, has no obvious cross reaction with non-associated proteins, and can be used as a key reagent for constructing a high-sensitivity and high-specificity detection kit; the detection accuracy of the MuSK protein can be remarkably improved, and the important clinical application value is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibodies, in particular, to an antibody against MuSK protein or related products and applications thereof. BACKGROUND

[0002] MuSK (Muscle-Specific Kinase) is a receptor tyrosine kinase that plays a crucial role in the formation, maintenance and function of the neuromuscular junction (NMJ). It is a single-pass transmembrane protein located on the muscle cell membrane, responsible for the clustering of AChR at the NMJ and the maintenance of the postsynaptic membrane. When the motor neuron releases agrin and other signaling molecules to bind to the extracellular domain of MuSK on the membrane to form dimers, the protein kinase function is activated to phosphorylate the tyrosine residues in the tail, i.e., to form a signal complex. The phosphorylated tyrosine becomes a binding site for intracellular signaling proteins, which expands the information and activates a series of biochemical reactions in the skeletal muscle cells, i.e., the signal transduction process. Like all tyrosine kinase receptors, the protein includes an extracellular region containing immunoglobulin (IgG)-like motifs, a transmembrane region, and an intracellular region. The extracellular domain includes three IgG-like domains and one cysteine-rich domain, and the intracellular region has tyrosine kinase activity to initiate downstream signaling pathways.

[0003] Dysfunction of MuSK protein is closely related to various diseases, among which the most prominent is myasthenia gravis (MG), an autoimmune disease caused by autoantibodies attacking components of the neuromuscular junction, characterized by muscle weakness and easy fatigue. About 10% of MG patients have anti-MuSK antibodies of IgG4 type, and these patients usually show severe muscle weakness, especially affecting the eye, face, throat and respiratory muscles, and have poor response to conventional acetylcholinesterase inhibitor therapy. In addition, mutations in the MuSK gene can also cause another genetic neuromuscular transmission disorder, congenital myasthenic syndrome, which is characterized by muscle weakness, respiratory and feeding difficulties in infants or children.

[0004] However, most of the commercially available MuSK antibodies are mouse / rabbit-derived monoclonal antibodies / polyc lonal antibodies, and there is a lack of high-quality anti-MuSK antibodies with excellent binding properties.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The present application aims to provide an antibody against MuSK protein or related products and applications thereof.

[0007] The present application is implemented as follows: In a first aspect, an embodiment of the present application provides an antibody or antigen binding fragment thereof against MuSK protein, comprising: LCDR1, LCDR2 and LCDR3 in a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 1 or 13, and HCDR1, HCDR2 and HCDR3 in a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 2 or 14.

[0008] In a second aspect, an embodiment of the present application provides an antibody conjugate, comprising: the antibody or antigen binding fragment thereof according to the foregoing embodiment.

[0009] In a third aspect, an embodiment of the present application provides a reagent or kit, comprising: the antibody or antigen binding fragment thereof according to the foregoing embodiment, or the antibody conjugate according to the foregoing embodiment.

[0010] In a fourth aspect, an embodiment of the present application provides use of the antibody or antigen binding fragment thereof according to the foregoing embodiment, or the antibody conjugate according to the foregoing embodiment, in the preparation of a product for detecting MuSK protein or MuSK protein detection for non-disease treatment or diagnosis purposes.

[0011] In a fifth aspect, an embodiment of the present application provides an isolated nucleic acid or a vector containing the nucleic acid, encoding the antibody or antigen binding fragment thereof according to the foregoing embodiment.

[0012] In a sixth aspect, an embodiment of the present application provides a recombinant cell containing the isolated nucleic acid or the vector containing the nucleic acid according to the foregoing embodiment.

[0013] In a seventh aspect, an embodiment of the present application provides a preparation method of the antibody or antigen binding fragment thereof according to the foregoing embodiment, comprising: culturing the recombinant cell according to the foregoing embodiment.

[0014] The present application has the following beneficial effects: The anti-MuSK antibody provided by the present application exhibits extremely high affinity and specificity, can accurately bind to specific antigen epitopes of MuSK protein, has no obvious cross-reaction with non-related proteins, and can be used as a key reagent to construct a high-sensitivity and high-specificity detection kit, which can significantly improve the detection accuracy of MuSK protein and has important clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0016] Figure 1 Purification results of MuSK protein in Example 1; Figure 2 Immunofluorescence results for detecting mouse antibody production in Example 1; Figure 3 Immunofluorescence screening results of positive hybridoma cells in Example 1; Figure 4 Four-parameter curve for identifying the affinity of anti-human MuSK human-mouse chimeric monoclonal antibody in Example 5; Figure 5 Resulting graph for identifying the specificity of anti-human MuSK human-mouse chimeric monoclonal antibody in Example 5; wherein, Figure 5 -a is the immunofluorescence result of the chimeric antibody on the LRP4 overexpression slide, Figure 5 -b is the immunofluorescence result of the chimeric antibody on the agrin overexpression slide; Figure 5 -c is the immunofluorescence result of the chimeric antibody on the MuSK overexpression slide; Figure 5 -d is the immunofluorescence result of the chimeric antibody incubated with 594-labeled goat anti-mouse secondary antibody; Figure 6 Staining comparison results of the chimeric antibody and patient serum; Figure 7 Expression results of the chimeric antibody for detecting MuSK protein on overexpression cell slide; Figure 8 Expression results of the chimeric antibody for detecting MuSK protein on gastrocnemius muscle. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0018] The term "antibody" in this paper is used in the broadest sense, which can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the desired biological activity, such as specific binding to MuSK antigen or its fragments.

[0019] The term "antigen-binding fragment" herein refers to a portion of an intact antibody that binds the same antigen as the intact antibody. Those skilled in the art will understand from the teachings of the present disclosure that antigen-binding fragments can be produced by methods known in the art, for example, by enzymatic digestion, including pepsin or papain, and / or by chemical reduction to break disulfide bonds, and can also be synthesized by recombinant genetics or by automated peptide synthesizers, such as the Applied BioSystems automated peptide synthesizer.

[0020] The term "CDR" herein refers to the "complementarity determining region" and refers to the highly variable regions of immunoglobulin heavy and light chains, which are involved in antigen binding and are the regions that contain the amino acid residues that form the binding site for the antigen or epitope recognized by the antibody or antigen-binding fragment.

[0021] The term "framework region" herein refers to the "skeleton region" or "FR" region of an antibody and refers to the regions of the variable region of the heavy chain that are outside the CDR regions; the heavy chain skeleton region can be further subdivided into regions adjacent to the CDRs (FR1, FR2, FR3, and FR4), wherein the heavy chain skeleton region can be further subdivided into regions adjacent to the CDRs, including HFR1, HFR2, HFR3, and HFR4 skeleton regions. The heavy chain variable region is obtained by arranging the CDRs and FRs (from the amino-terminal end to the carboxy-terminal end) in the following order: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0022] The term "percent identity" herein refers to the extent to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned. Alignment of amino acid sequence percent identity can be performed in various ways known in the art, such as the software known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA, etc. Those skilled in the art can determine appropriate parameters for aligning sequences.

[0023] In one aspect, the embodiments of the present application provide an antibody or antigen-binding fragment thereof against MuSK protein, which comprises: LCDR1, LCDR2, and LCDR3 in the light chain variable region of the amino acid sequence shown in SEQ ID NO: 1 or 13, and HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 2 or 14.

[0024] In optional embodiments, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are defined by any one of the Kabat, Chothia, IMGT, AbM, or Contact systems.

[0025] In optional embodiments, the antibody or antigen-binding fragment thereof comprises: an LCDR1 of the amino acid sequence set forth in SEQ ID NO: 3, an LCDR2 of the amino acid sequence set forth in SEQ ID NO: 4, and an LCDR3 of the amino acid sequence set forth in SEQ ID NO: 5 or 6; and an HCDR1 of the amino acid sequence set forth in SEQ ID NO: 7, an HCDR2 of the amino acid sequence set forth in SEQ ID NO: 8, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO: 9 or 10.

[0026] In optional embodiments, the heavy chain variable region and the light chain variable region further comprise a framework region.

[0027] In optional embodiments, the antibody or antigen-binding fragment thereof comprises: a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 1 or 13, and a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 2 or 14.

[0028] In optional embodiments, the antibody or antigen-binding fragment thereof further comprises a constant region.

[0029] In optional embodiments, the constant region is of bovine, equine, porcine, ovine, caprine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human origin.

[0030] In optional embodiments, the constant region comprises a heavy chain constant region and a light chain constant region.

[0031] In optional embodiments, the heavy chain constant region is selected from the heavy chain constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; and the light chain constant region is selected from a kappa-type or lambda-type light chain constant region.

[0032] In optional embodiments, the heavy chain constant region has an amino acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 18; and the light chain constant region has an amino acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 17.

[0033] In optional embodiments, the antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv, and scFv of the antibody.

[0034] In another aspect, embodiments of the present application provide an antibody conjugate comprising: the antibody or antigen binding fragment thereof of any of the preceding embodiments; In optional embodiments, the antibody conjugate further comprises a label, a purification tag and / or a solid support conjugated to the antibody or antigen binding fragment thereof.

[0035] In optional embodiments, the label comprises a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.

[0036] In optional embodiments, the solid support comprises, but is not limited to, a microsphere, a plate, and a membrane.

[0037] In optional embodiments, the solid support comprises any one or more of a magnetic microsphere, a plastic microsphere, a plastic microparticle, a latex microsphere, a microplate, glass, a capillary, nylon, and a nitrocellulose membrane.

[0038] In another aspect, embodiments of the present application provide a reagent or a kit comprising: the antibody or antigen binding fragment thereof of any of the preceding embodiments or the antibody conjugate of any of the preceding embodiments.

[0039] In another aspect, embodiments of the present application provide use of the antibody or antigen binding fragment thereof of any of the preceding embodiments or the antibody conjugate of any of the preceding embodiments in the manufacture of a medicament for detecting MuSK protein or for non-disease treatment or diagnostic purposes of MuSK protein detection. In optional embodiments, the sample for detection can comprise any one or more of an environmental sample, a biological sample, and an artificially prepared positive sample. The biological sample comprises, but is not limited to, plasma, whole blood, serum, tissue, cells, and extracts of cells and extracts of tissue cells.

[0040] In another aspect, embodiments of the present application provide an isolated nucleic acid or a vector comprising the nucleic acid, which encodes the antibody or antigen binding fragment thereof of any of the preceding embodiments.

[0041] In optional embodiments, the nucleic acid sequence encoding the light chain variable region of SEQ ID NO: 1 is set forth in SEQ ID NO: 11.

[0042] In optional embodiments, the nucleic acid sequence encoding the heavy chain variable region of SEQ ID NO: 2 is set forth in SEQ ID NO: 12.

[0043] In optional embodiments, the nucleic acid sequence encoding the light chain variable region of SEQ ID NO: 13 is set forth in SEQ ID NO: 15.

[0044] In an alternative embodiment, the nucleic acid sequence encoding the heavy chain variable region of SEQ ID NO: 14 is set forth in SEQ ID NO: 16.

[0045] In an alternative embodiment, the vector comprising the nucleic acid comprises a recombinant expression vector. The backbone vector of the recombinant expression vector comprises a plasmid or a viral vector; as another embodiment, the backbone vector of the recombinant expression vector can be a plasmid vector; as another embodiment, the plasmid vector is one or more of, but not limited to, pcDNA3.1, pBAD, pQE-12, pGEX, pBluescript, pET-series expression vectors, pCAI-n, pPOW3.0, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, pREP, pCEP4, pMC1neo, pXT1, pSG5, EBO-pSV2neo, pBPV-1, pFUSE, pRSVgpt, pRSVneo, pIZD35, pRc / CMV, pcDNA1, pcDNA3.1, pSPORT1, pGEMHE, pLXIN, pSIR, pIRES-EGFP, pEAK-10, pTriEx-Hygro, pCINeo, pAO815, pPIC9K, and pPIC3.5K; in a particular embodiment, the backbone vector of the recombinant expression vector is pcDNA3.1.

[0046] In another aspect, embodiments of the present application provide a recombinant cell comprising the isolated nucleic acid or the vector comprising the nucleic acid of any of the preceding embodiments.

[0047] In an alternative embodiment, the recombinant cell comprises a prokaryotic cell or a eukaryotic cell; the prokaryotic cell comprises, but is not limited to, a bacterial cell such as E. coli; the eukaryotic cell comprises, but is not limited to, a yeast cell, an insect cell, an animal cell, or a plant cell; the yeast cell can be, but is not limited to, a Pichia or a Saccharomyces cell; the animal cell can be, but is not limited to, a CHO cell, a COS cell, a NSO cell, a 293T cell, a HT-1080 cell, a BHK (baby hamster kidney cell), a HEK (human embryonic kidney cell), an Expi293F, or a PERC.6 (human retinal cell).

[0048] In an alternative embodiment, the way of introducing the vector into the recombinant cell comprises a physical method, a chemical method or a biological method; as another implementation, the physical method comprises calcium phosphate precipitation, lipofection, particle bombardment, microinjection or electroporation; as another implementation, the chemical method comprises a colloidal dispersion system or a lipid-based system; as another implementation, the biological method comprises a DNA vector, a lentivirus vector, a poxvirus vector, a herpes simplex virus vector, an adenovirus vector or an adeno-associated virus vector.

[0049] In addition, the embodiments of the present application further provide a preparation method of the antibody or the antigen binding fragment thereof according to any of the foregoing embodiments, which comprises culturing the recombinant cell according to any of the foregoing embodiments.

[0050] The features and properties of the present application are further described in detail below in combination with the embodiments.

[0051] Example 1 Preparation of mouse anti-human MuSK monoclonal antibody 1.1 Preparation of MuSK recombinant protein The sequence of the human MuSK gene was obtained from the GenBank database, and a 6his tag sequence was added to the C-terminal of the gene. The human MuSK gene sequence was cloned into a pET28a vector by the GenScript Corporation, and a plasmid was constructed and named pET28a-6his. MuSK The sequence of the human MuSK gene was obtained from the GenBank database, and a 6his tag sequence was added to the C-terminal of the gene. The human MuSK gene sequence was cloned into a pET28a vector by the GenScript Corporation, and a plasmid was constructed and named pET28a-6his. MuSK The sequence of the human MuSK gene was obtained from the GenBank database, and a 6his tag sequence was added to the C-terminal of the gene. The human MuSK gene sequence was cloned into a pET28a vector by the GenScript Corporation, and a plasmid was constructed and named pET28a-6his. MuSK The sequence of the human MuSK gene was obtained from the GenBank database, and a 6his tag sequence was added to the C-terminal of the gene. The human MuSK gene sequence was cloned into a pET28a vector by the GenScript Corporation, and a plasmid was constructed and named pET28a-6his. 600 The sequence of the human MuSK gene was obtained from the GenBank database, and a 6his tag sequence was added to the C-terminal of the gene. The human MuSK gene sequence was cloned into a pET28a vector by the GenScript Corporation, and a plasmid was constructed and named pET28a-6his. Figure 1 The sequence of the human MuSK gene was obtained from the GenBank database, and a 6his tag sequence was added to the C-terminal of the gene. The human MuSK gene sequence was cloned into a pET28a vector by the GenScript Corporation, and a plasmid was constructed and named pET28a-6his.

[0052] 1.2 Animal immunization Six 6-8 week old female Balb / c mice were immunized with the MuSK-6his recombinant protein prepared above, with an antigen dosage of 40 μg per mouse. For the first immunization, 40 μg of antigen was mixed with an equal volume of Freund's adjuvant and thoroughly ground into a water-in-oil emulsion. 100 μL (containing 40 μg of antigen) of the mixture was then injected intraperitoneally. Two weeks after the first immunization, a second immunization was administered, with 40 μg of antigen mixed 1:1 with incomplete Freund's adjuvant, and 100 μL (containing 40 μg of antigen) of the mixture was injected intraperitoneally. Two weeks after the second immunization, a third immunization was administered, using the same dosage, method, and route of antigen injection as the second immunization. Two weeks later, a booster immunization was given via intraperitoneal injection of the antigen. Three days later, tail blood was collected for immunofluorescence detection of antibody production.

[0053] 1.3 Immunofluorescence detection of antibodies produced by mice Serum from six immunized mice was collected and incubated with MuSK-overexpressing cell slices. Based on immunofluorescence results, mice corresponding to serum with the highest specificity for MuSK-overexpressing cell slices were selected for hybridoma cell fusion. Preparation of MuSK-overexpressing cell slices: Using molecular biology methods, [the following steps were taken]... MuSK The gene was ligated into pCDNA3.1 to obtain the recombinant plasmid pCDNA3.1- MuSK The recombinant plasmid was transfected into 293T cells with a cell density of 30%–50% using PEI transfection reagent (the transfection reagent was purchased from Thermo Biotechnology and transfection was performed according to the instructions). After 48 hours of transfection, methanol was added for fixation for 5 minutes, followed by washing twice with PBS. After drying, the cell slices were cut and set aside. The empty pCDNA3.1 control slices were prepared in the same manner.

[0054] Immunofluorescence assay procedure: Serum from six immunized mice was diluted 1:100 and 1:500 using PBST buffer, and incubated with MuSK overexpressing cell smears at room temperature for 1 hour. The cells were washed three times with PBST buffer, 5 minutes each time. A 1:200 dilution of Alexa Fluor 594-labeled goat anti-mouse IgG secondary antibody (Jackson) was used, and the cells were incubated at room temperature for 30 minutes. The cells were washed three times with PBST buffer, 5 minutes each time. Results were observed under a 20x fluorescence microscope. Figure 2 It can be seen that, compared with the serum results of unimmunized normal mice, the serum of mice No. 2 and No. 4 showed the strongest fluorescence signal, indicating that their immunization results were most specific to the MuSK protein. Mice No. 2 and No. 4 were selected for subsequent experiments.

[0055] 1.4 Cell fusion and hybridoma screening Cell fusion: macrophages were taken from common mouse ascites for culture, and after 2 days of culture, they were used as feeder cells; the 2nd and 4th mice after immunization were sacrificed by cervical dislocation, and were disinfected in 75% ethanol. The spleen cells of the immunized mice were taken out, cut and ground to obtain a single spleen cell suspension. The mouse spleen cells and the myeloma cells SP2 / 0 in the logarithmic growth phase were fused at a ratio of 10:1 at 37°C in a water bath. The supernatant was removed by centrifugation at 500g for 10 min, and then the cells were resuspended with DMEM medium containing HAT, and were dispensed into 96-well cell culture plates with feeder cells, 100 μL / well, and then the culture plates were incubated at 37°C in a 5% CO2 incubator. The next day after fusion, 100 μL of HAT culture solution was added to each well, and then HAT culture solution was added once every 2-3 days, and screening was performed after about 2 weeks of culture.

[0056] Hybridoma screening: the growth of hybridoma cells was observed, and after 7-10 days, when the cell culture supernatant turned yellow, an appropriate amount of cell supernatant was taken for immunofluorescence detection. The cell supernatant in each row of 12 wells of the 96-well plate was mixed in equal volumes, and according to the immunofluorescence results, the mixed sample of 19F, 22D and 23F with strong positive signal was selected for further culture, and the parent clone was further screened. After 7-10 days of continuous culture, the three rows of mixed samples with strong positive signals were detected by immunofluorescence detection of antibodies in each well of the cell supernatant, respectively, and finally 19F8, 22D8 and 23F2 were screened as positive wells. The positive wells screened were changed to verify again, and were subjected to limiting dilution to culture about 1 cell per well. The cell culture supernatant was taken for immunofluorescence detection, and the results are shown in Figure 3

[0057] Example 2 Identification of mouse anti-human MuSK monoclonal antibody ​2.1 Antibody titer detection: The purified MuSK-6his protein was re-coated as an antigen to detect the titer of 22D8-m mouse monoclonal antibody. The antigen coating concentration was 100 ng / well, and the coating was performed at 4°C overnight. The next day, PBST was used to wash 3 times, each for 3 min, and then dried. The monoclonal 22D8 cell supernatant was diluted by 3 times, 100 μL / well, and 4# mouse tail blood was used as a positive control at 1:200, and normal mouse serum was used as a negative control at 1:200, 100 μL / well, and incubated at 37°C for 1 h. PBST was used to wash 3 times, each for 3 min, and then dried. HRP-labeled secondary antibody was used, the antibody was diluted at 1:5000 (manufacturer: Jackson), and incubated at 37°C for 30 min. PBST was used to wash 3 times, each for 3 min, and then dried. TMB was added for color development for 5 min, H2SO4 was used to terminate color development, and the absorbance value was measured at 450 nm. The detection results are shown in Table 1.

[0058] Table 1. Detection results

[0059] 2.2 Antibody typing identification: The purified MuSK-6his protein was re-coated as an antigen to detect the typing of 22D8-m mouse monoclonal antibody. The antigen coating concentration was 100 ng / well, and the coating was performed at 4°C overnight. The next day, PBST was used to wash 3 times, each for 3 min, and then dried. The monoclonal 22D8 cell supernatant was added at 100 μL / well, and 4# mouse tail blood was used as a positive control at 1:200, 100 μL / well, and incubated at 37°C for 1 h. PBST was used to wash 3 times, each for 3 min, and then dried. HRP-labeled secondary antibody (total IgG / IgG1 / IgG2a / IgG2b / IgG3 / IgM / Ig Kappa chain / Ig lambda chain) was used, the antibody was diluted at 1:5000 (manufacturer: Jackson), and incubated at 37°C for 30 min. PBST was used to wash 3 times, each for 3 min, and then dried. TMB was added for color development for 5 min, H2SO4 was used to terminate color development, and the absorbance value was measured at 450 nm. The experimental results are shown in Table 2, and the heavy chain of 22D8-m mouse monoclonal antibody is IgG1, and the light chain is Ig Kappa chain. Then, the 22D8-m mouse monoclonal antibody was purified from the culture supernatant of positive hybridoma cells, and the purified antibody sample was used for subsequent antibody detection and functional experiments.

[0060] Table 2. Experimental results

[0061] 2.3 Heavy chain and light chain variable region sequencing The 22D8 hybridoma cells screened for large-scale culture were lysed by Trizol, and total RNA of the hybridoma cell lysate was extracted. The RNA was separated by agarose gel electrophoresis, and the first strand cDNA was synthesized by 5' RACE using the RNA as a template. The hybridoma cell monoclonal antibody heavy chain variable region (VH) gene and light chain variable region (VL) gene were amplified by PCR using the first strand cDNA as a template. The double-strand cDNA of the antibody light chain / heavy chain variable region was extracted and sequenced by a sequencing company to obtain the light chain variable region amino acid sequence (SEQ ID NO: 1) and the heavy chain variable region amino acid sequence (SEQ ID NO: 2) of the 22D8-m murine monoclonal antibody. The CDR region of the 22D8-m murine monoclonal antibody light chain (wherein the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO: 3, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO: 4, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO: 5) and the CDR region of the heavy chain amino acid sequence (wherein the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO: 7, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO: 8, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO: 9) were labeled using the Kabat method.

[0062] Example 3 Construction and expression of human-mouse chimeric monoclonal antibody recombinant vector 3.1 Design of human-mouse chimeric monoclonal antibody light chain and heavy chain sequences against human MuSK The antibody sequence was amplified using FastPfu DNA Polymerase by a conventional method of molecular biology, and a mutant of the antibody sequence was constructed. The amino acid A at position 117 of the light chain variable region amino acid sequence of the 22D8-m murine monoclonal antibody shown in SEQ ID NO: 1 was mutated to T, and the new amino acid sequence after mutation was SEQ ID NO: 13, and the corresponding nucleotide sequence was changed from SEQ ID NO: 11 to SEQ ID NO: 15. The amino acid Y at position 126 of the heavy chain variable region amino acid sequence of the 22D8-m murine monoclonal antibody shown in SEQ ID NO: 2 was mutated to F, and the new amino acid sequence after mutation was SEQ ID NO: 14, and the corresponding nucleotide sequence was changed from SEQ ID NO: 12 to SEQ ID NO: 16.

[0063] The light chain of the designed anti-human MuSK human-mouse chimeric monoclonal antibody is composed of a mouse-derived light chain variable region (SEQ ID NO: 1 or SEQ ID NO: 13) and a human kappa type light chain constant region (SEQ ID NO: 17), and the heavy chain is composed of a mouse-derived heavy chain variable region (SEQ ID NO: 2 or SEQ ID NO: 14) and a human IgG4 heavy chain constant region (SEQ ID NO: 18). The specific anti-human MuSK human-mouse chimeric monoclonal antibodies obtained have the following four combinations, as shown in Table 3, and are named 4 groups of antibodies as 22D8-h1, 22D8-h2, 22D8-h3 and 22D8-h4.

[0064] Table 3 Anti-human MuSK human-mouse chimeric monoclonal antibody combinations

[0065] 4.2 Construction and expression and purification of recombinant vectors of anti-human MuSK human-mouse chimeric monoclonal antibodies The nucleotide sequence of the mouse-derived monoclonal antibody light chain variable region (SEQ ID NO: 11 and SEQ ID NO: 15) was amplified, and the human kappa chain constant region nucleotide sequence (SEQ ID NO: 19) was amplified. The light chain variable region and the light chain constant region fragments were connected to the vector pcDNA3.1 using homologous recombination, and the variable region amplification fragment and the constant region amplification fragment were connected in the order of 5'-3' direction, and the insertion position was the multiple cloning site of the vector pcDNA3.1, and the two groups of recombinant vectors obtained were labeled as pcDNA3.1- MuSK -VL and pcDNA3.1- MuSK -VL'; and the nucleotide sequence of the mouse-derived monoclonal antibody heavy chain variable region (SEQ ID NO: 12 and SEQ ID NO: 16) was amplified, and the human IgG4 heavy chain constant region sequence (SEQ ID NO: 20) was amplified. The two fragments were connected to the vector pcDNA3.1 using homologous recombination, and the variable region amplification fragment and the constant region amplification fragment were connected in the order of 5'-3' direction, and the insertion position was the multiple cloning site of the vector pcDNA3.1, and was labeled as pcDNA3.1- MuSK -VH and pcDNA3.1- MuSK -VH'. The connected recombinant plasmid was sent to Shengong Biotechnology for sequencing, and the sequenced recombinant plasmid was large-scale extracted and used for cell transfection to prepare antibodies.

[0066] The following four recombinant vectors were co-transfected into Expi293F cells using the transfection reagent PEI for transient expression. The supernatant was collected 3 days after transfection, purified using protein A packing material, and the protein concentration was determined using the BCA kit to obtain four designed anti-human MuSK human-mouse chimeric monoclonal antibodies.

[0067] Table 4. Anti-human MuSK chimeric monoclonal antibodies

[0068] Example 5: Application of anti-human MuSK human-mouse chimeric monoclonal antibody 5.1 ELISA validation of the affinity of the anti-MuSK human-mouse chimeric antibody The purified MuSK protein was coated overnight at 4°C using carbonate buffer (pH 9.6), 100 ng / well. The next day, the wells were washed three times with PBST for 3 min each time, and then patted dry. The wells were then blocked with 2% BSA and incubated at 37°C for 1 h. The wells were washed three times with PBST for 3 min each time, and then patted dry. The concentration of the four groups of MuSK human-mouse chimeric antibodies obtained in Example 4 was adjusted to 5 µg / mL, and then serially diluted 5-fold. 100 µL of diluted antibody was added to each well and incubated at 37°C for 1 h. The wells were washed three times with PBST for 3 min each time, and then patted dry. The corresponding goat anti-human IgG-HRP secondary antibody diluted 1:5000 was incubated at 37°C for 30 min. The wells were washed three times with PBST and then patted dry. TMB was used for color development for 10 min, and the reaction was stopped with 2 M H2SO4. The absorbance values ​​measured at 450 nm are shown in Table 5.

[0069] Table 5 Absorbance values

[0070] Plot a four-parameter curve based on ELISA values. Figure 4 The EC50 values ​​of the four antibody groups were calculated. The results showed that, ranked by EC50 value, 22D8-h3 (EC50=18.046) < 22D8-h4 (EC50=19.422) < 22D8-h1 (EC50=19.490) < 22D8-h2 (EC50=20.978). All four groups of MuSK human-mouse chimeric antibodies obtained in this invention exhibited good affinity.

[0071] 5.2 Immunofluorescence verification of the specificity of the anti-MuSK human-mouse chimeric antibody a. Verification of the specificity of anti-MuSK human-mouse chimeric antibody using different antigens: LRP4 (NM_002334.4) / agrin (NM_001305275.2) overexpression crawlers were prepared according to the method in Example 1. These two overexpression crawlers were combined with the MuSK overexpression crawler to form a detection chip. Four groups of anti-human MuSK human-mouse chimeric antibodies were diluted 1:100 and incubated onto the detection chip at room temperature for 1 hour. The chips were washed three times with PBST for 5 minutes each time. FITC-labeled goat anti-human IgG4 diluted 1:200 was added, and the chips were incubated at room temperature for 30 minutes. The chips were washed three times with PBST buffer for 5 minutes each time. The results were observed under a fluorescence microscope. The results are shown below (…). Figure 5 -a、 Figure 5 -b and Figure 5 As shown in -c).

[0072] b. Verification of the specificity of anti-MuSK human-mouse chimeric antibodies using secondary antibodies of different species: Four groups of anti-MuSK human-mouse chimeric antibodies were diluted 1:100 and incubated with MuSK overexpression cell smears at room temperature for 1 h. The cells were washed three times with PBST for 5 min each time. Then, 1:200 diluted 594-labeled goat anti-mouse IgG4 was added, and the cells were incubated at room temperature for 30 min. The cells were washed three times with PBST buffer for 5 min each time. The results were observed under a fluorescence microscope. Figure 5 -d).

[0073] Compare( Figure 5 -a、 Figure 5 -b and Figure 5 -c) Results showed that the four groups of anti-human MuSK chimeric antibodies reacted only with the MuSK overexpression chip and not with other antigens on the chip, indicating that the anti-human MuSK chimeric antibodies prepared in this invention can specifically recognize the MuSK antigen and have good specificity; compared ( Figure 5 -c and Figure 5 -d) Results: The four groups of anti-human MuSK human-mouse chimeric antibodies showed obvious green fluorescence signals only when incubated with FITC-labeled goat anti-human IgG4 secondary antibody, while no obvious red fluorescence signal was observed when incubated with 594-labeled goat anti-mouse IgG4 secondary antibody. This indicates that the four groups of chimeric antibodies only react with anti-human secondary antibody and not with anti-mouse secondary antibody, demonstrating that the four groups of human-mouse chimeric antibodies modified in this invention have good species specificity.

[0074] 5.3 Immunofluorescence verification of the function of the anti-MuSK human-mouse chimeric antibody Adjust the concentration of 4 groups of anti-human MuSK human-mouse chimeric antibodies and 22D8-m mouse monoclonal antibodies to 100 μg / mL, and dilute 4 groups of anti-human MuSK human-mouse chimeric antibodies and 22D8-m mouse monoclonal antibodies with PBST solution at a volume ratio of 1:100, dilute commercial MuSK antibody (Santa: sc-134398, 100 μg / ml) 1:100, dilute MuSK antibody positive patient serum 1:10, respectively, incubate with MuSK overexpression cell slides and pcDNA3.1 control slides, incubate at room temperature for 1 h, wash with PBST for 3 times, 5 min each time, add 1:200 diluted FITC labeled goat anti-human / mouse IgG4, incubate at room temperature for 30 min, wash with PBST buffer for 3 times, 5 min each time; observe the results under a fluorescence microscope at 20 times magnification.

[0075] According to Figure 6 The results show that the 4 groups of anti-human MuSK human-mouse chimeric antibodies can be incubated with the same secondary antibody under the same conditions as the patient samples (the labeled secondary antibody is FITC labeled goat anti-human IgG4), and the typical morphology of the positive cells is consistent, with more obvious humanization species advantage, which can be used as a substitute for antibody positive serum and play a positive reference role in related applications; and compared with the mouse monoclonal antibody before modification, the immunofluorescence signal intensity is basically the same, which proves that the activity of the 4 groups of anti-human MuSK human-mouse chimeric antibodies after modification is basically the same as that of the mouse monoclonal antibody before modification, which can accurately identify MuSK protein and can also be used for other scientific research, with very good application value and very important scientific research guiding significance.

[0076] Example 6 Fluorescein conjugated anti-human MuSK protein human-mouse chimeric antibody Fluorescein isothiocyanate (FITC) conjugated anti-human MuSK protein human-mouse chimeric antibody can be used to detect the expression and distribution of MuSK protein in tissue or cell samples. Take 22D8-h3 monoclonal antibody as an example for labeling, the steps are as follows: Material preparation: fluorescent reagent: FITC reagent; buffer: PBS, pH 7.4, coupling buffer: 0.05 mol / L carbonate buffer (CBS, pH 9.0-9.5); consumables: dialysis bag S1. Antibody pretreatment: dialyze the antibody to the coupling buffer, and the concentration of the antibody is 1 mg / mL; S2. Dissolve the FITC reagent with anhydrous DMSO to a final concentration of 1 mg / mL, vortex well, and then stand at room temperature for 5 min to completely dissolve (avoid ultrasonic, to prevent FITC degradation); S3. Add the FITC reagent to the antibody solution slowly according to the molar ratio of FITC: antibody = 10:1, and react at room temperature for 30 minutes-2 hours (or overnight at 4°C) in the dark; S4. Add 1 / 10 volume of 1 M glycine (pH 7.0) or Tris-HCl (pH 8.0) to terminate the reaction, and incubate for 10-30 minutes; S5. Dialysis in PBS (pH 7.4) at 4°C for 24 hours (3-4 times of buffer exchange) to remove free FITC; S6. Sterile filtration of the dialyzed labeled antibody, take a small sample to test OD 280 and OD 495, calculate the concentration: labeled antibody concentration = (A280-0.35 x A495) / antibody extinction coefficient IgG about 1.35), finally get the concentration of FITC-labeled anti-human MuSK protein human-mouse chimeric antibody about 0.6 mg / mL; S7. Store at 4°C in the dark.

[0077] Example 7: Kit for detecting MuSK protein and its application 1. Kit 1 for detecting MuSK protein and its application Kit 1 components: FITC-conjugated anti-human MuSK protein human-mouse chimeric monoclonal antibody 22D8-h3, detection buffer, washing buffer and anti-fluorescence quencher. Among them, the FITC-conjugated anti-human MuSK protein human-mouse chimeric monoclonal antibody is the core component of the kit.

[0078] Principle and application of kit 1: The kit uses the principle of immunofluorescence, uses the FITC-conjugated anti-human MuSK protein human-mouse chimeric monoclonal antibody to detect MuSK protein in the tissue or cell sample to be tested. After the antigen-antibody reaction, a complex carrying green fluorescein will be formed, which will emit bright green fluorescence under a fluorescence microscope, so as to accurately determine the expression, position of MuSK protein, and carry out corresponding localization, qualitative or quantitative analysis.

[0079] Method for using kit 1: Referring to Example 1, three batches of MuSK overexpression cell slides were prepared, and the FITC-conjugated anti-human MuSK protein human-mouse chimeric monoclonal antibody 22D8-h3 in Example 6 was diluted 1:50 using the detection buffer for incubation, and then washed 3 times with the washing buffer. After adding the anti-fluorescence quencher, the results were observed under a fluorescence microscope. According to the fluorescence results, it can be judged that MuSK protein is expressed on the three batches of cell slides. Figure 7

[0080] 2. Kit 2 for detecting MuSK protein and its application ​The kit consists of two components: human-mouse chimeric monoclonal antibody 22D8-h4 against human MuSK protein, 4% paraformaldehyde fixative, 0.3% Triton X-100 permeabilization buffer, 5% BSA blocking buffer, antibody dilution buffer, wash buffer, Alexa Fluor 594-labeled anti-human secondary antibody, and anti-fluorescence quencher. The human-mouse chimeric monoclonal antibody against human MuSK protein is the core component of this kit.

[0081] Kit 2 Principle and Application: This kit uses the principle of indirect immunofluorescence. First, a human-mouse chimeric monoclonal antibody against human MuSK protein specifically binds to the MuSK antigen in the tissue or cell to be tested. Then, an anti-human secondary antibody labeled with Alexa Fluor 594 binds to the specific antibody to form an antigen-specific antibody-labeled fluorescent antibody complex, thereby realizing the expression characteristics and subcellular localization detection of MuSK protein in tissues or cells.

[0082] Kit 2 Instructions: Take frozen sections of rat gastrocnemius muscle, thaw them, fix them with 4% paraformaldehyde (PFA) for 30 min, wash three times with wash buffer, treat with 0.3% Triton X-100 permeabilization buffer at room temperature for 30 min, block with 5% BSA for 1 h, dilute the human-mouse chimeric monoclonal antibody 22D8-h4 against human MuSK protein 1:10, add it to the sections, and incubate at room temperature for 1 h. Wash three times with wash buffer, add Alexa Fluor 594-labeled anti-human secondary antibody and incubate for 30 min, then read the slides under a fluorescence microscope.

[0083] according to Figure 8 It can be seen that the Musk protein is expressed in the gastrocnemius muscle of rats.

[0084] The sequence information involved in this application is as follows.

[0085]

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against a MuSK protein, characterized in that, It comprises: a LCDR1, a LCDR2 and a LCDR3 in a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 1 or 13 and a HCDR1, a HCDR2 and a HCDR3 in a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 2 or 14.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The LCDR1, the LCDR2, the LCDR3, the HCDR1, the HCDR2 and the HCDR3 are defined by any one of Kabat, Chothia, IMGT, AbM or Contact system; Optionally, the antibody or the antigen binding fragment thereof comprises: a LCDR1 having an amino acid sequence as shown in SEQ ID NO: 3, a LCDR2 having an amino acid sequence as shown in SEQ ID NO: 4 and a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 5 or 6; and a HCDR1 having an amino acid sequence as shown in SEQ ID NO: 7, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 8 and a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 9 or 10.

3. The antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein, The antibody or the antigen binding fragment thereof further comprises a constant region; Optionally, the species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the constant region comprises a heavy chain constant region and a light chain constant region; Optionally, the heavy chain constant region is selected from a heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD or a combination of multiple constant regions; the light chain constant region is selected from a kappa type or lambda type light chain constant region; Optionally, the amino acid sequence of the heavy chain constant region has at least 80% identity with the sequence shown in SEQ ID NO: 18; the amino acid sequence of the light chain constant region has at least 80% identity with the sequence shown in SEQ ID NO:

17.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein, The antigen binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

5. An antibody conjugate, characterized in that, It comprises: The antibody or the antigen binding fragment thereof of any one of claims 1-4; Optionally, the antibody conjugate further comprises a label, a purification tag and / or a solid phase carrier coupled to the antibody or the antigen binding fragment thereof.

6. A reagent or kit characterized in that, It comprises: The antibody or the antigen binding fragment thereof of any one of claims 1-4 or the antibody conjugate of claim 5.

7. Use of the antibody or the antigen binding fragment thereof of any one of claims 1-4 or the antibody conjugate of claim 6 in the manufacture of a product for detecting MuSK protein or for non-disease treatment or diagnosis purpose of MuSK protein detection.

8. An isolated nucleic acid or a vector containing said nucleic acid, characterized in that, It encodes the antibody or the antigen binding fragment thereof of any one of claims 1-4.

9. A recombinant cell, characterized in that, It contains the isolated nucleic acid of claim 8 or a vector containing the nucleic acid.

10. The method of producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein It comprises: Culturing the recombinant cell of claim 9.

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