Fusion protein and use thereof

Abstract

The present invention provides a fusion protein for diagnosing myasthenia gravis, which detects AChR autoantibodies with high sensitivity and specificity, thus enabling rapid and accurate diagnosis of myasthenia gravis.

Description

Fusion proteins and their uses

[0001] The present invention relates to a fusion protein and its use.

[0002] Myasthenia gravis is a disease in which muscles easily tire due to abnormalities in muscle control through nerve stimulation. It is caused by a malfunction in parasympathetic nerve signal transmission through acetylcholine at the neuromuscular junction (NMJ). Myasthenia gravis is an autoimmune disease involving autoantibodies. Autoantibodies to acetylcholine receptors (AChR), muscle specific kinase (MuSK), and LDL receptor related protein 4 (LPR4) present in the postsynaptic neuromuscular synapse can be detected in the blood and muscle tissue of patients. Among these, autoantibodies to acetylcholine receptors can be found in more than 80% of patients. Myasthenia gravis initially presents as ocular myasthenia gravis, with mild symptoms such as ptosis, a temporary drooping of the eyelids. When the disease progresses to generalized myasthenia gravis, muscle movement becomes difficult, causing difficulties in daily life. If it worsens, it can lead to death due to the inability to control the respiratory and circulatory muscles. About 20% of patients with myasthenia gravis experience a myasthenic crisis, which is a condition in which they experience difficulty breathing and speech, within two years after diagnosis. If this is not treated properly, it can lead to death. This type of myasthenia gravis can be diagnosed relatively easily through characteristic clinical features such as muscle weakness and fatigue without sensory or other neurological deficits, and the typical location of muscle weakness and preservation of tendon reflexes.To diagnose myasthenia gravis, repetitive nerve stimulation test, which provides electrical stimulation to specific muscles and measures muscle contraction responses, and single fiber electromyography (SEM), which measures the electrical activity of individual muscle fibers within the muscle, are performed. Since abnormalities are observed in the thymus in approximately 75% of patients, chest computed tomography is necessary. In this regard, the following prior art provides information including the pathophysiology, differential diagnosis, treatment, diagnostic precision examination, patient education, and a diagnostic algorithm for ptosis and weakness associated with myasthenia gravis (Shannon MA, et al., J. Am. Acad. Nurse. Pract. 15(2):72-8. 2003).

[0003] Myasthenia gravis is caused by various autoantibodies against acetylcholine receptors, muscle-specific kinase, and LDL receptor-associated protein 4, and the autoantibodies found induce the disease through different pathological mechanisms. Therefore, treatment methods may vary depending on the type of autoantibody found. For example, myasthenia gravis patients found to have muscle-specific kinase-specific autoantibodies in their blood may expect high efficacy when treated with rituximab, which depletes B cells. However, patients found to have autoantibodies against acetylcholine receptors show low efficacy. Therefore, distinguishing which autoantibodies a patient has may be important for appropriate treatment of myasthenia gravis.

[0004] Radioimmunoassay is used to detect autoantibodies against acetylcholine receptors (AChRs), the most common antigens found in patients with myasthenia gravis. This method involves reacting patient samples and serum with radiolabeled AChR proteins, and then detecting AChR autoantibodies through a non-competitive binding method using immunoprecipitation. This method can determine whether a patient is AChR-positive or -negative. However, current methods for measuring AChR autoantibodies cannot distinguish between autoantibodies that are pathogenic and those that simply bind to AChRs without any pathological effects. In fact, the amount of AChR-binding autoantibodies measured using current diagnostic methods does not correlate with the severity of the patient's disease. This may be due to the inability to distinguish between antibodies that are actually pathogenic. Therefore, a diagnostic method that can determine the presence or absence of pathogenic antibodies in the blood or specimens of patients with myasthenia gravis, and further, the degree of pathogenic activity of autoantibodies in the blood, can be useful in predicting the severity of the patient's disease and selecting an appropriate treatment method.

[0005] The present invention provides a method for detecting acetylcholine autoantibodies causing pathological effects in a sample or blood of a patient with myasthenia gravis and measuring the degree of pathological effects.

[0006] However, although the above-mentioned repetitive nerve stimulation test and single fiber electromyography test have high sensitivity, they may show normal findings in some patients, and the initial symptoms are nonspecific, which often delays diagnosis. In addition, they require skilled examiners and special equipment, which makes them difficult to access.

[0007] The present invention aims to solve various problems, including the above-mentioned problems, and provides a fusion protein for diagnosing myasthenia gravis, which can rapidly and accurately diagnose myasthenia gravis by detecting AChR autoantibodies with high sensitivity and specificity, and its use. However, these tasks are exemplary and the scope of the present invention is not limited thereby.

[0008] According to one aspect of the present invention, a fusion protein is provided in which the extracellular domain of a nicotinic acetylcholine receptor unit is linked to the N-terminus of the transmembrane / cytosolic domain of a homotypic pentameric membrane protein excluding the acetylcholine receptor.

[0009] According to another aspect of the present invention, an artificial acetylcholine receptor is provided, which is produced by pentamerization of the above fusion protein and has a ligand binding function of the acetylcholine receptor.

[0010] According to another aspect of the present invention, a polynucleotide encoding the fusion protein is provided.

[0011] According to another aspect of the present invention, a recombinant vector comprising the polynucleotide is provided.

[0012] According to another aspect of the present invention, a transformed cell into which the recombinant vector has been introduced is provided.

[0013] According to another aspect of the present invention, a composition for diagnosing myasthenia gravis is provided, comprising the transformed cell as an active ingredient.

[0014] According to another aspect of the present invention, there is provided a method for treating a transformed cell with an acetylcholine receptor-specific autoantibody and serum of a subject;

[0015] A step of confirming the pathological activity of the transformed cells and acetylcholine receptor-specific autoantibodies; and

[0016] A method for providing information for diagnosing and treating myasthenia gravis through detection of acetylcholine receptor-specific autoantibodies in the serum of a subject is provided, which comprises a step of confirming the severity of myasthenia gravis and determining a treatment method in a subject who exhibits similar pathological activity to a positive control group treated with human serum containing acetylcholine receptor autoantibodies and significantly higher pathological activity than a negative control group treated with normal serum.

[0017] The fusion protein for diagnosing myasthenia gravis of the present invention, as described above, can be usefully used to measure the pathological activity of AChR autoantibodies appearing in myasthenia gravis patients due to its unique high sensitivity and specificity, thereby helping to select appropriate treatment methods and predict prognosis. In addition, it can be utilized in the development of an immunotherapy agent for treating myasthenia gravis through the production of a chimeric autoantibody receptor (CAR). Of course, the scope of the present invention is not limited by these effects.

[0018] FIG. 1 is a schematic diagram schematically showing the structure of a fusion protein of AChRα1β1δε manufactured according to one embodiment of the present invention.

[0019] Figure 2 is ChR-5-HT3 of the present invention. A This graph shows the results of analyzing the cell surface expression of the fusion protein.

[0020] Figure 3: AChR-5-HT3 A This graph shows the results of analyzing the formation of membrane attack complexes by autoantibodies after production of expression cell lines.

[0021] Figure 4 AChR-5-HT3 AThis graph shows the results of analyzing the sensitivity to AChR autoantibodies using expression cell lines.

[0022] Figure 5 AChR-5-HT3 A This graph shows the results of analyzing complement-dependent apoptosis by autoantibodies using expression cell lines.

[0023] Figure 6: AChR-5-HT3 A This graph shows the results of analyzing complement-dependent cell death according to the type of autoantibody using expression cell lines.

[0024] Figure 7 AChR-5-HT3 A This graph shows the results of analyzing AChR internalization according to autoantibody type using expression cell lines.

[0025] Figure 8 AChR-5-HT3 in human T cells A This is a FACs graph showing the results of analyzing the expression of mRNA by introducing AChRα-5-HT3 in PBMCs stimulated with CD3 / CD28 beads. A CAAR constructs fused with the CD28 transmembrane domain, 4-1BB co-stimulatory domain, CD3z, and AChRβ1-5-HT3 genes A , AChRδ-5-HT3 A , AChRε-5-HT3 A Four types of mRNA were simultaneously introduced by electroporation, and 24 hours later, AChR on the surface of T cells was detected with autoantibodies and bungarotoxin.

[0026] Figure 9 is a FACs graph showing the results of analyzing whether autoantibody binding occurs due to fetal AChR (alpha1, beta, delta, and gamma) expression.

[0027] Figure 10 is a FACs graph showing the results of analyzing whether autoantibody binding occurs due to the expression of Alpha1-HT3A.

[0028] Definition of terms:

[0029] The term "myasthenia gravis" used in this document is a disease caused by a disorder of neurotransmission at the neuromuscular junction, and its main symptoms are fluctuating muscle weakness and muscle fatigue. The characteristic symptoms are fluctuating muscle weakness and muscle fatigue, which occur because the number of acetylcholine receptors at the neuromuscular junction decreases. Although the exact cause of myasthenia gravis is not yet known, the pathogenesis is known to be an antibody-mediated autoimmune disease. In other words, the mechanism is known to be that antibodies are produced in the body against acetylcholine receptors present on the surface of muscles at the neuromuscular junction, where the nerves connect to the muscles, and the antibody binds to the receptor, preventing the binding of the neurotransmitter acetylcholine, thereby deteriorating the function of the neuromuscular junction.

[0030] The term "acetylcholine receptor (AChR)" used in this document refers to a protein receptor that plays a crucial role in the nervous system. When the neurotransmitter acetylcholine binds to this receptor, various physiological responses occur. Acetylcholine receptors are broadly divided into nicotinic acetylcholine receptors (nAChR) and muscarinic acetylcholine receptors (mAChR).

[0031] The term "autoantibody" as used in this document refers to an antibody (a type of protein) produced by the immune system in response to a specific component of one's own body. These autoantibodies are responsible for a variety of autoimmune diseases.

[0032] The term "complement activation" used in this document refers to the process by which complement proteins, which play a crucial role in the human immune system, are activated to eliminate pathogens and induce an inflammatory response. Complement proteins are present in the blood and are activated when infection or inflammation occurs, triggering processes such as lysis, which destroys and kills the cell membranes of pathogens such as bacteria and viruses, and phagocytosis, which coats the surface of pathogens to make them readily engulfed by phagocytes. There are three main complement activation pathways: the classical pathway, the alternative pathway, and the lectin pathway.

[0033] Detailed description of the invention:

[0034] According to one aspect of the present invention, a fusion protein is provided in which the extracellular domain of a nicotinic acetylcholine receptor unit is linked to the N-terminus of the transmembrane / cytosolic domain of a homotypic pentameric membrane protein excluding the acetylcholine receptor.

[0035] In the above fusion protein, the membrane protein forming the homotypic pentamer may be a membrane protein capable of forming a homotypic pentamer among ligand-gated ion channels. Specifically, the membrane protein forming the homotypic pentamer may be selected from the group consisting of a serotonin receptor including a 5-HT3 homotypic pentamer, a GABAA receptor, an Erwinia chyrysanthemi ligand-gated ion channel (ELIC), a glycine receptor, a Gloeobacter violaceus ligand-gated ion channel (GLIC), a formate-nitrite transporter (FNT), and a Mycobacterium tuberculosis mechanosensitive channel of large conductance (MscL).

[0036] In the above fusion protein, the homotypic pentameric membrane protein is 5-HT3 A , 5-HT3 B , 5-HT3 C , 5-HT3 D and 5-HT3 E 5-HT3 receptors selected from the group consisting of β1, β1S265N, β2, β3, ρ1, ρ2 and ρ3; GABA selected from the group consisting of AA receptor, an Erwinia chyrysanthemi ligand-gated ion channel (ELIC), a glycine receptor selected from the group consisting of α1, α2, α3 and α4, a Gloeobacter violaceus Ligand-Gated Ion Channel (GLIC), a formate-nitrite transporter (FNT) and a Mycobacterium tuberculosis mechanosensitive channel of large conductance (MscL), which may be selected from the group consisting of the 5-HT3 A The transmembrane / cytoplasmic domain may comprise an amino acid sequence represented by SEQ ID NO: 12.

[0037] The above nicotinic acetylcholine receptor may be a heteropentameric muscle nAChR, and the heteropentameric muscle nAChR is composed of four subunits, AChRα1, AChRβ1, AChRδ, and AChRε, wherein the heteropentamer is composed of five subunits, namely, two α1s, one β1, one δ, and one ε. In addition, for AChRα1 and AChRβ1, other isotypes may be used, and such isotypes include AChRα2, AChRα3, AChRα4, AChRα5, AChRα6, AChRβ2, AChRβ2, AChRβ3, and AChRβ4. Such isotypes may also be used in the present invention. In a preferred embodiment of the fusion protein of the present invention, the acetylcholine receptor subunit used is AChRα1, AChRβ1, AChRδ, or AChRε. However, since AChRε is an adult acetylcholine receptor subunit, and the AChRγ subunit is used instead of AChRε during the fetal period, AChRγ can be used instead of AChRε in the fusion protein of the present invention.

[0038] The above fusion protein may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 to 28. At this time, the fusion protein may additionally have a tag peptide or epitope peptide linked to the N-terminus for ease of detection or separation and purification, and the tag or epitope peptide may be an HA tag, a 6XHis tag, a Myc tag, a GST-tag, or a FLAG tag.

[0039] In the above fusion protein, the nicotinic acetylcholine receptor unit may be a fetal form AChR selected from the group consisting of AChRα1, AChRβ1, AChRδ, and AChRγ, or an adult form AChR selected from the group consisting of AChRα1, AChRβ1, AChRδ, and AChRε.

[0040] According to another aspect of the present invention, an artificial acetylcholine receptor is provided, which is produced by pentamerization of the fusion protein and has a ligand binding function of an acetylcholine receptor. The pentamerization of the fusion protein is produced by heterotypic pentamerization of a fusion protein comprising AChRα1, AChRβ1, AChRδ, and AChRε, respectively, or by heterotypic pentamerization of a fusion protein comprising AChRα1, AChRβ1, AChRδ, and AChRγ, respectively, wherein the ratio of AChRα1, AChRβ1, AChRδ, and AChRε may be 2:1:1:1, or the ratio of AChRα1, AChRβ1, AChRδ, and AChRγ may be 2:1:1:1.

[0041] In the artificial acetylcholine receptor, the extracellular domain of AChRα1 may include an amino acid sequence represented by SEQ ID NO: 7, the extracellular domain of AChRβ1 may include an amino acid sequence represented by SEQ ID NO: 8, the extracellular domain of AChRδ may include an amino acid sequence represented by SEQ ID NO: 9, the extracellular domain of AChRε may include an amino acid sequence represented by SEQ ID NO: 10, and the extracellular domain of AChRγ may include an amino acid sequence represented by SEQ ID NO: 11.

[0042] According to another aspect of the present invention, a polynucleotide encoding the fusion protein is provided.

[0043] The above polynucleotide may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 29 to 33.

[0044] According to another aspect of the present invention, a recombinant vector comprising the polynucleotide is provided.

[0045] In the above recombinant vector, the polynucleotide may be included in the form of a gene construct operably linked to a regulatory sequence.

[0046] The term "operably linked to" as used herein means that the desired nucleic acid sequence (e.g., in an in vitro transcription / translation system or in a host cell) is linked to the regulatory sequence in such a way that its expression can occur.

[0047] The term "regulatory sequence" above includes promoters, enhancers, and other regulatory elements (e.g., polyadenylation signals). Regulatory sequences include those that direct the constant expression of a target nucleic acid in many host cells, those that direct the expression of a target nucleic acid only in specific tissue cells (e.g., tissue-specific regulatory sequences), and those that direct expression to be induced by a specific signal (e.g., inducible regulatory sequences). It will be understood by those skilled in the art that the design of an expression vector may vary depending on factors such as the choice of host cell to be transformed and the level of protein expression desired. The expression vector of the present invention can be introduced into a host cell to express the fusion protein. Regulatory sequences that enable expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, these typically include regulatory sequences responsible for transcription initiation and, optionally, a poly-A signal responsible for transcription termination and stabilization of the transcript. In addition to transcriptional regulators, additional regulatory sequences may include translational enhancers and / or native or heterologous promoter regions. For example, potential regulatory sequences that allow expression in mammalian host cells include the CMV-HSV thymidine kinase promoter, SV40, RSV (Rowe's sarcoma virus) promoter, human elongation factor 1α-promoter, glucocorticoid-inducible MMTV (Moloney mouse tumor virus) promoter, metallothionein-inducible or tetracycline-inducible promoters, or enhancers such as the CMV enhancer or SV40 enhancer. For expression in neuronal cells, it is contemplated that the neurofilament promoter, PGDF promoter, NSE promoter, PrP promoter, or thy-1 promoter may be used. The above promoters are known in the art and are described in the literature (Charron, J.et al., Biol. Chem. 270: 25739-25745, 1995).For expression in prokaryotic cells, a number of promoters have been disclosed, including the lac promoter, the tac promoter, or the trp promoter. In addition to factors capable of initiating transcription, the regulatory sequences may also include a transcription termination signal, such as the SV40-poly-A site or the TK-poly-A site, downstream of the polynucleotide according to one embodiment of the present invention. Suitable expression vectors in the present invention are known in the art, including, for example, the Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCO BRL), pGX-27 (Patent No. 1442254), pX (Pagano et al., Science. 255: 1144-1147, 1992), yeast two-hybrid vectors such as pEG202 and dpJG4-5 (Gyuris et al., Cell, 75: 791-803, 1995) or prokaryotic expression vectors such as lambda gt11 or pGEX (Amersham Pharmacia). In addition to the nucleic acid molecules of the present invention, the vector may further comprise a polynucleotide encoding a secretion signal. The above secretion signals are well known to those skilled in the art. Furthermore, depending on the expression system used, a leader sequence capable of directing the recombinant protein to a cellular compartment is combined with the coding sequence of the polynucleotide according to one embodiment of the present invention, preferably a leader sequence capable of directly secreting the translated protein or its protein into the cytoplasmic periphery or extracellular medium.

[0048] In addition, the vector of the present invention can be produced, for example, by standard recombinant DNA techniques, which include, for example, blunt-end and sticky-end ligation, restriction enzyme treatment to provide appropriate termini, removal of phosphate groups by alkaline phosphatase treatment to prevent inappropriate joining, and enzymatic ligation by T4 DNA ligase. The vector of the present invention can be produced by recombining DNA encoding a signal peptide obtained by chemical synthesis or genetic recombination technology, DNA encoding a recombinant fusion protein of the present invention or a fusion protein comprising the same into a vector containing an appropriate regulatory sequence. The vector containing the above regulatory sequence can be purchased or produced commercially.

[0049] The above expression vector may additionally include a polynucleotide encoding a signal sequence, and the signal sequence is used for presentation of the recombinant protein expressed in the cell to the cell surface via the endoplasmic reticulum -> Golgi apparatus, and the signal sequences of various transmembrane proteins may be used, and in the present invention, the signal sequence of the acetylcholine receptor itself to be expressed is used as an example, but is not limited thereto.

[0050] The expression vector according to one embodiment of the present invention may be an expression vector capable of expressing the protein in a host cell, and the expression vector may take any form, such as a plasmid vector, a viral vector, a cosmid vector, a phagemid vector, an artificial human chromosome, etc.

[0051] According to another aspect of the present invention, a transformed cell into which the recombinant vector has been introduced is provided.

[0052] The above transformed cell can be any prokaryotic or eukaryotic cell as a host cell. As for the prokaryotic cell, any bacteria or archaea such as gram-positive or gram-negative bacteria can be used, and as for the eukaryotic cell, any mammalian cell, insect cell, or fungus such as yeast can also be used, but more preferably, a mammalian cell such as a human cell is used.

[0053] In the above-mentioned transformed cells, an artificial acetylcholine receptor can be expressed on the cell surface by introducing AChRα1, AChRβ1, AChRδ and AChRε in a ratio of 2:1:1:1 or introducing AChRα1, AChRβ1, AChRδ and AChRγ in a ratio of 2:1:1:1, and the transformed cells may be those in which a gene encoding a complement inhibitor is knocked out, and the complement inhibitor may be CD45, CD55 and CD59.

[0054] The above-mentioned transformed cell comprises an expression vector (for convenience, referred to as a 'first expression vector') including a polynucleotide (for convenience, referred to as a 'first polynucleotide') encoding a fusion protein to which the transmembrane / cytoplasmic domains of an AChRα1 ECD-homopentamer-forming protein are linked, an expression vector (for convenience, referred to as a 'second expression vector') including a polynucleotide (for convenience, referred to as a 'second polynucleotide') encoding a fusion protein to which the transmembrane / cytoplasmic domains of an AChRβ1 ECD-homopentamer-forming protein are linked, an expression vector (for convenience, referred to as a 'third expression vector') including a polynucleotide (for convenience, referred to as a 'third polynucleotide') encoding a fusion protein to which the transmembrane / cytoplasmic domains of an AChRδ ECD-homopentamer-forming protein are linked, and a fusion protein to which the transmembrane / cytoplasmic domains of an AChRε ECD-homopentamer-forming protein are linked or AChRγ It is possible to produce it by cotransfecting a host cell with an expression vector (for convenience, referred to as the 'fourth expression vector') containing a polynucleotide (for convenience, referred to as the 'fourth polynucleotide') encoding a fusion protein in which the transmembrane / cytoplasmic domains of an ECD-homotype pentameric protein are linked at a ratio of 2:1:1:1. Alternatively, it can be produced by inserting the first to fourth polynucleotides at an appropriate ratio into one or more vectors and then transforming the host cell so that these polynucleotides are transfected at the above-described ratio of 2:1:1:1. In this case, in order to insert two or more polynucleotides into one expression vector, an expression vector is used in which two or more gene cassettes having multiple regulatory sequences necessary for gene expression, such as two or more promoters, enhancers, and poly A, are inserted in two or more places, or two or more polynucleotides are linked to an IRES (internal ribosome entry site) behind one regulatory sequence, thereby enabling expression in the form of a polycistron.

[0055] According to another aspect of the present invention, a composition for diagnosing myasthenia gravis is provided, comprising the transformed cell as an active ingredient.

[0056] In the above diagnostic composition, the myasthenia gravis can be selected from the group consisting of ophthalmic, bulbar, generalized, neonatal transient, congenital persistent, and familial infantile. The ophthalmic type is a type in which symptoms appear only in the eye muscles, and the main symptoms are ptosis and diplopia, and accounts for about 15% of all myasthenia gravis patients. The bulbar type shows symptoms such as difficulty speaking, swallowing, and breathing due to weakness of the oral, pharyngeal, and laryngeal muscles, and can progress to the generalized type. The generalized type is the most common type in which weakness appears not only in the eye muscles but also in the muscles of the entire body, including the limbs and trunk, and accounts for about 85% of all myasthenia gravis patients. The neonatal transient type is a type that appears temporarily when the mother's myasthenia gravis antibodies are transferred to the fetus, and in most cases, the symptoms improve within a few weeks after birth. Congenital persistent myasthenia gravis is a very rare form of myasthenia gravis that persists from birth due to genetic factors. Familial infantile myasthenia gravis is a rare form of myasthenia gravis that develops in infancy due to genetic factors.

[0057] According to another aspect of the present invention, there is provided a method for treating a transformed cell with an acetylcholine receptor-specific autoantibody and serum of a subject;

[0058] A step of confirming the pathological activity of the transformed cells and acetylcholine receptor-specific autoantibodies; and

[0059] A method for providing information for diagnosing and treating myasthenia gravis through detection of acetylcholine receptor-specific autoantibodies in the serum of a subject is provided, which comprises a step of confirming the severity of myasthenia gravis and determining a treatment method in a subject who exhibits similar pathological activity to a positive control group treated with human serum containing acetylcholine receptor autoantibodies and significantly higher pathological activity than a negative control group treated with normal serum.

[0060] In the above information providing method, the step of confirming whether the transformed cell binds to the acetylcholine receptor-specific autoantibody may be performed by fluorescence-activated cell sorting (FACS) analysis, analysis of whether the acetylcholine receptor-specific autoantibody is internalized into cells, or complement-dependent cytotoxicity (CDC) analysis by complement activation. In this case, in the complement-dependent cytotoxicity analysis, the transformed cell line according to one embodiment of the present invention may additionally have a gene encoding a complement inhibitor knocked out, and the complement inhibitor may be CD45, CD55, and CD59.

[0061] The term "diagnosis" as used in this document includes all types of analyses used to predict the likelihood of developing a disease or to determine or derive the risk of developing a disease.

[0062] Approximately 85% of patients with myasthenia gravis have AChR autoantibodies, and complement activation and receptor internalization at the NMJ due to AChR autoantibodies are known to be the main causes of the pathology in patients with myasthenia gravis. For the treatment of myasthenia gravis, acetylcholinesterase inhibitors are prescribed for patients with mild symptoms, and immunosuppressants and steroids are used later depending on the severity of the symptoms. Antibody therapy such as Soliris and Rituxan are also prescribed to prevent acute exacerbations and manage the disease. In emergency situations or when symptoms are severe, plasma exchange or intravenous immunoglobulin (IVIG) injections are used, and thymectomy may be performed in cases where a thymic tumor is present.

[0063] Effective treatment for myasthenia gravis may vary depending on the target antigen of the autoantibody. For patients with autoantibodies to muscle-specific kinase (MuSK), B-cell depletion therapies like Rituxan are more effective than those with autoantibodies to the AChR. Conversely, complement inhibitors like Soliris (eculizumab) and Ultomiris (ravulizumab) are effective in controlling disease in patients with AChR autoantibodies, but their efficacy is reduced in patients with MuSK autoantibodies. This is because each autoantibody has a different pathogenesis depending on its target substance. Therefore, identifying the presence and type of autoantibody in a patient is a crucial diagnostic factor in patient treatment. However, the current diagnostic method for autoantibodies in myasthenia gravis (MG) patients only detects antibodies that bind to each target protein. While numerous types of antibodies exist in patients' blood, and some of these antibodies may bind to target substances, not all of them are responsible for the patient's symptoms. The target binding affinity and binding location of autoantibodies vary depending on the type of antibody, and autoantibodies present in myasthenia gravis patients are typically a mixture of polyclonal antibodies derived from various B cells. Furthermore, the correlation between the amount of target-binding autoantibodies present in a patient's blood and the severity of the disease is unclear. Therefore, measuring the total amount of target-binding antibodies has limitations in predicting the patient's symptoms. If only a portion of target-binding autoantibodies play a significant role in the pathogenesis of the disease, while the remaining antibodies have minimal effects, a strong correlation between the amount of autoantibodies and disease severity is unlikely. Therefore, the detection of antibodies that are crucial for the pathogenesis of the disease may be more important than the presence or abundance of target-binding antibodies.Therefore, a technology that directly measures the pathological activity of autoantibodies could be very useful in overcoming the limitations of current autoantibody diagnostic methods.

[0064] Autoantibodies can contribute to the pathogenesis of myasthenia gravis in different ways depending on their structure and target. The autoantibodies that cause myasthenia gravis vary depending on their target. Furthermore, the process by which autoantibody-producing B cells are generated also varies, which affects the pathogenesis and treatment. Patients with autoantibodies to MuSK and LRP4 do not develop thymoma, and the autoantibodies are mostly IgG4. On the other hand, patients with autoantibodies to AChR are associated with the development of thymoma, and the autoantibodies are IgG1. IgG1 and IgG4 differ in their receptor binding and activation, which leads to different immune responses. AChR autoantibodies detected in a significant number of myasthenia gravis patients are IgG1, which can induce complement activation through binding to C1q and other mechanisms. It is known that AChR autoantibodies cause pathological effects by activating complement at the NMJ, and therefore complement inhibitors such as Soliris and Ultomiris are effective in controlling the disease. However, MuSK and LRP4 are IgG4 antibodies that cannot activate complement. In addition to complement activation, AChR autoantibodies are thought to cause pathological effects through receptor internalization and interference with ACh binding. Therefore, the molecular mechanisms of pathological effects may differ depending on the type of autoantibody.

[0065] Meanwhile, the acetylcholine receptor is a receptor protein to which the neurotransmitter acetylcholine binds, and is composed of an ion channel receptor. It is mainly distributed in the neuromuscular junction and autonomic ganglia and plays an important role in the nervous and muscular systems. The acetylcholine receptor is largely divided into nicotinic acetylcholine receptor (nAChR) and muscarinic acetylcholine receptor (mAChR). The nAChR is an ion channel receptor that responds to nicotine and is mainly distributed in the neuromuscular junction and autonomic ganglia and when acetylcholine is bound, Na + , K + , Ca 2+ It opens ion channels of the back and is involved in nerve transmission and muscle contraction. In addition, the mAChR is a G protein-coupled receptor that responds to muscarinic receptors and is mainly distributed in postganglionic nerve fibers of the parasympathetic nervous system. When acetylcholine is bound, it activates intracellular signaling pathways and is involved in controlling heart rate and digestive functions.

[0066] It has been reported that 5,000 to 10,000 acetylcholine receptors (nAChRs) are present specifically in the muscle fiber cell membrane of the neuromuscular junction (NMJ) where the muscle fiber and nerve are connected. The width of the NMJ is 1 μm. 2Since the acetylcholine receptor is only present in a very high density in that area, acetylcholine receptors exist in a very high density. Therefore, when autoantibodies against acetylcholine receptors are produced, the density of autoantibodies binding to acetylcholine receptors at the NMJ can become very high. This high density of autoantibody-antigen complexes creates a favorable environment for complement activation. In particular, the C1q protein, which is abundant in the blood, binds to 5-6 antibody molecules at once, inducing the formation of a membrane attack complex (MAC) by complement activation in the cell membrane. Therefore, the situation in which autoantibodies bind in large quantities to the target AChR in a narrow area creates a more suitable environment for complement activation. When MAC is formed, the cell membrane is damaged, causing cell death or loss of function. Complement activation and NMJ damage by acetylcholine receptor autoantibodies are one of the major causes of myasthenia gravis. It is quite difficult to reproduce the NMJ environment containing a high density of acetylcholine receptors in cell lines that are easy to culture. To artificially form NMJs in vitro, myoblasts must be induced to differentiate into myotubes, which in turn differentiate into motor neurons, and then co-cultured to facilitate NMJ formation. However, this method requires prolonged cell differentiation, making it difficult to achieve high differentiation efficiencies across experiments. Furthermore, differentiated cells are difficult to maintain due to their inability to grow.

[0067] The heteropentamer muscle nicotinic acetylcholine receptor, which is mainly present in adults, is formed by a complex of five proteins (α2βδε) expressed from four genes. To more easily express the acetylcholine receptor, a method can be considered to express the acetylcholine receptor in a cell line that is easy to culture. However, even if all four genes are introduced into cells, the efficiency of the proteins expressing and forming a normal complex on the cell membrane is very low. When a chaperone protein such as the receptor-associated protein of the synapse (rapsyn) that helps complex formation is coexpressed, an increase in the cell membrane expression of the acetylcholine receptor can be observed. However, even in this case, expression is observed only in a portion of cells. Introduction of genes such as rapsyn alone has limitations in inducing high density of acetylcholine receptors on the cell membrane at the level of the NMJ. If high-density acetylcholine receptors are not expressed, complement activation is difficult to occur even if antibodies bind, making it impossible to reproduce complement activation by autoantibodies in patients with myasthenia gravis.

[0068] Internalization of acetylcholine receptors (AChRs) induced by autoantibodies can also cause pathological reactions by reducing the amount of AChRs present on the cell surface. Crosslinks between AChRs and autoantibodies may be crucial for receptor internalization. Therefore, internalization may require binding of the pathogenic antibody to a high density of AChRs formed at the NMJ. If it were possible to develop a cell line that is easy to culture and proliferate and expresses a high density of AChRs on the cell membrane, this could overcome the limitations of current technologies that only detect and quantify AChR autoantibodies and contribute to the development of cell-based assays capable of measuring the pathological activity of AChR autoantibodies in the blood. These assays could then be utilized to help patients with myasthenia gravis select more appropriate treatment options. Additionally, if acetylcholine receptors can be easily expressed on the cell membrane of immune cells including T cells, they can be used to develop immune cell therapy for treating myasthenia gravis by producing chimeric autoantibody receptors (CARs).

[0069] Previously, to diagnose myasthenia gravis, repetitive nerve stimulation tests were used to measure muscle contraction responses by electrically stimulating specific muscles, single-fiber electromyography tests to measure the electrical activity of muscle fibers, and acetylcholine receptor antibody tests to detect autoantibodies to acetylcholine receptors in the blood. However, these diagnostic prior technologies had problems such as low accessibility, as some patients showed normal findings, myasthenia gravis had nonspecific initial symptoms, which often delayed diagnosis, and skilled examiners and special equipment were required.

[0070] Accordingly, the present inventors developed a fusion protein for diagnosing myasthenia gravis that can express a large amount of AChR autoantibody binding sites on the cell surface. Using a transformed cell line containing the fusion protein, FACS analysis, acetylcholine receptor internalization analysis, and CDC were used to analyze the binding of receptors and ligands. As a result, complement-dependent apoptosis by the autoantibody was observed to occur specifically in the transformed cell line, and it was confirmed that AChR autoantibodies can induce AChR internalization. Therefore, the fusion protein of the present invention can measure complement activation and receptor internalization by autoantibodies and can measure the pathological action of AChR autoantibodies, and thus can be usefully utilized for measuring the pathological activity of AChR autoantibodies appearing in myasthenia gravis patients (Fig. 1).

[0071] Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the examples disclosed below, but can be implemented in various different forms. The following examples are provided to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention.

[0072] Example 1: Preparation of human acetylcholine receptor (AchR) expressing cell line

[0073] To prepare a human AchR expressing cell line, the present inventors transduced 293T cells one day before transduction using 0.5x10 6Cells were seeded into 6-well culture plates at 2 mL RPMI (10% FBS, 1X anti-anti) / well and cultured in a CO2 incubator. The next day, the culture medium was removed, and a mixture of human acetylcholine receptor subunit viruses A1:B:D:E was mixed in a ratio of 2:1:1:1, and mixed with 2 mL of medium. The mixture was treated with the cells, and polybrene was added at 1 μg / mL and cultured in a CO2 incubator. The next day, the culture medium was removed again, replaced with 2 mL of medium, and 1x10 cells were grown for cell expansion and sorting. 7 The number of cells exceeding 10 cells was secured. The transduced 293T cells were separated using EDTA, stained with AO / PI, and counted using a cell counter. The number of cells was 1x10 6 Cells were stained with 1 μg of mAb637 per cell at 4°C for 15 minutes. After washing, the cells were stained with anti-human Alexa 647 at 4°C for 15 minutes, and the mAb637-binding population was sorted using a cell sorter. The above method was repeated approximately twice and sorted to obtain cells highly expressing human acetylcholine receptor. Information on the reagents used to prepare the cell line is summarized in Table 1 below.

[0074] Reagent InformationReagent NameManufacturerCat NoLot NoDulbecco's Modified Eagle's Medium (DMEM)WelgeneLM001-05LM01200905FBSGibco26140-0792134834Anti-Anti (100x)Gibco15240-0622199836Trypsin-EDTAGibco25200-0562470031Dulbecco's Phosphate-Buffered Saline (D-PBS) (1X), liquidWelgeneLB001-02LB01201002BSASigmaA3294-100GSLBX1181Alexa Fluor 647 AffiniPure Goat Anti-Human IgGJacksonImmunoResearch109-605-00615-2370PolybreneSigmaD2438-50MLRNBK5603mAb637in house production20210706

[0075] Example 2: Internalization assay

[0076] Internalization assay is an experimental technique that measures how well antibodies or proteins enter cells, and can be used to determine the efficiency of drug delivery into cells. For the internalization assay, the inventors placed 1x10 cells in a 96-well plate the day before the assay. 4293T-AchR cells were seeded per well. The following day, the medium was removed, and mAb637, mAb192, and mAb35 antibodies (produced in house) conjugated with Fabflour-pH red (Satorius, Cat# 4722) at a molar ratio of 1:3 for 15 min at 37°C were treated in duplicate at 4 or 10 μg / ml. After incubation for 24 h at 37°C, internalization was monitored at 30-min intervals using Incucyte, and the red fluorescence resulting from antibody internalization was analyzed using the Incucyte analysis program. Information on the reagents used in the internalization assay is summarized in Table 2 below.

[0077] Reagent InformationReagent NameManufacturerCat NoLot NoFabflour-pH redSatorius47222311001213FBSGibco26140-0792134834Anti-Anti (100x)Gibco15240-062219983696 Well Cell Culture PlateNunc167008171226Trypsin-EDTAGIBCO252000562186972mAb637Made in house20210706mAb35Made in house20211220mAb192Made in house20231122

[0078] Example 3: Binding assay

[0079] For binding assays, 293T-AchR cells were detached using 10 mM EDTA and seeded at 1x10 in 96-well plates. 5Cells were dispensed per well. The cells were treated with 8 μg / ml of acetylcholine antibody and incubated at 4°C for 20 minutes to allow antigen binding. Subsequently, anti-human Alexa 647 antibody was treated at a 1:1000 concentration in FACs buffer and incubated at 4°C for 20 minutes. After staining and washing, the degree of acetylcholine receptor-specific antibody binding was analyzed by FACs analysis. Information on the reagents used in the binding assay is summarized in Table 3 below.

[0080] Reagent InformationReagent NameManufacturerCat NoLot NoDulbecco's Modified Eagle's Medium(DMEM)WelgeneLM001-05LM01200905FBSGibco26140-0792134834Anti-Anti (100x)Gibco15240-062219983696 well U-bottom plateThermo Scientific163320BB0F29A341960.5 M EDTAThermo Scientific15694 500ML4312384Dulbecco's Phosphate-Buffered Saline (D-PBS) (1X),liquidWelgeneLB001-02LB01201002BSASigmaA3294-100GSLBX1181Alexa Fluor 647 AffiniPure Goat Anti-Human IgGJackson ImmunoResearch109-605-00615-2370mAb637in house manufactured20210706mAb35in house manufactured20211220mAb192in house manufactured20231122mAb587in house manufactured20220103Alpha-Bungarotoxin Alexa647InvitrogenB354502286292

[0081] Example 4: CDC assay

[0082] The complement-dependent cytotoxicity (CDC) assay performed in the present invention used Lenti X-293T cells expressing AChR-ECM via a lentiviral system and knocking out three complement inhibitors (CD45, CD55, CD59) using the CRSPR / Cas9 system. The cells were detached using EDTA one day before the experiment and seeded in a 48-well plate at a density of 1x10 per well. 5 Cells were seeded and cultured for one day. The next day, 10% normal human serum and antibodies were added to provide complement, and CDC was induced by incubating at 37°C for 6 hours. Anti-C5b-9 antibody was added at a ratio of 1:200 and incubated at 4°C for 15 minutes. After washing, secondary antibodies and FVD450 were added at a ratio of 1:100 and incubated at 4°C for 15 minutes. The percentage of dead cells or C9-positive cells was measured using FACs. Information on the reagents used in the CDC analysis is summarized in Table 4 below.

[0083] Reagent InformationReagent NameManufacturerCat NoLot NoPooled Human Complement SerumInnovative reserchICSER50ML3933748 well cell culture plateNunc15068760668069DMEMWelgeneLM001-05LM01211305FBSGIBCO26140-0792134834PBSWelgeneLB001-02LB01201402Trypsi n-EDTAGIBCO252000562186972AOPINexelomBioscienceCS2-0106-5ML191108-02-02L-GlutamineGIBCO25030-0812193033mAb637in house production20210706mAb637in house Production 20220103Anti-C5b-9 + C5b-8 antibody [aE11]Abcamab667681019750-4Mouse IgG2A Alexa Fluor 647R&D systemsIC003RAELD0322031eBioscience TM Fixable Viability Dye eFluor TM 450Invitrogen65-0863-142254157-R

[0084] Example 5: Preparation of fusion protein

[0085] Acetylcholine receptors, which are mainly present in adults, are formed by a complex of five proteins (α2βδε) expressed from four genes. Even when AChRα1, AChRβ1, AChRδ, and AChRε genes are introduced to express AChR on the surface of a cell line that is easy to proliferate, only a small amount of AChR is detected on the cell surface. Therefore, the inventors of the present invention have introduced the extracellular domain portion of AChRα1, AChRβ1, AChRδ, and AChRε and the 5-HT3, a serotonin receptor that forms a homo-pentamer on the cell surface, to overexpress AChR on the cell surface. AGenes that are fused with the transmembrane / cytosolic domain of the gene (AChRα1-5-HT3) A , AChRβ1-5-HT3 A , AChRδ-5-HT3 A , AChRε-5-HT3 A ) was manufactured (Fig. 1).

[0086] Example 6: Measurement of AChR cell surface expression

[0087] The present inventors have prepared the above AChRα1-5-HT3 A , AChRβ1-5-HT3 A , AChRδ-5-HT3 A , AChRε-5-HT3 A Introduction of four genes into 293T cells (AChR-5-HT3) A ) and then measured AChR cell surface expression by flow cytometry using fluorescently labeled AChR-binding bungarotoxin and various types of AChR-binding autoantibodies (mAb35, mAb587, mAb192, mAb637). As a result, expression was detected in almost all cells, and the expression level was also high (Fig. 2).

[0088] Example 7: Selection of AChR-5-HT3A expressing cell lines

[0089] The present inventors have AChRα1-5-HT3 A , AChRβ1-5-HT3 A , AChRδ-5-HT3 A , and AChRε-5-HT3 ATo produce a cell line that stably expresses four genes, the genes were introduced into the 293T cell line at a ratio of 2:1:1:1, and then flow cytometry analysis was performed using mAb637, an AChR-binding autoantibody, and Alexa 647, a secondary antibody. AchR expression was confirmed by increased mAb637 binding in cells expressing AchR compared to cells that did not express AchR (Fig. 3). After that, single cells were isolated and treated simultaneously with mAb637, an AChR autoantibody, and human normal serum. C5-b9 antibody was used to confirm whether a membrane attack complex was formed on the cell surface, and a single cell line with high sensitivity for the formation of a cell surface membrane attack complex when treated with AChR autoantibodies was selected through this (Fig. 4).

[0090] Example 8: Confirmation of complement-dependent cell death by autoantibodies

[0091] AChR-5-HT3 manufactured according to one embodiment of the present invention A When overexpressing cell lines were treated with AChR autoantibodies and human normal serum, cell death induced by complement activation was observed. AChR-5-HT3 A We observed that apoptosis occurred specifically in the overexpressing cell line (Fig. 5). Different types of AChR autoantibodies may have different pathological activities, such as complement activation, after binding. mAb637, an AChR autoantibody that showed pathological activity when administered to primates, was used to treat AChR-5-HT3 A Complement activity was observed well after treatment with cell lines. However, in the case of mAb587, binding to AChR was confirmed, but AChR-5-HT3 A No increase in cell death due to complement activation was observed when treated with cell lines (Fig. 6). These results indicate that AChR-5-HT3 AThe use of expression cell lines allowed the distinction between antibodies that bind to AChR but do not induce complement activation and AChR autoantibodies that can activate complement.

[0092] Example 9: Measurement of AChR internalization according to autoantibody type

[0093] In addition to complement activation, AChR autoantibodies are predicted to induce AChR internalization into cells, thereby lowering the concentration of AChR on the NMJ cell surface and causing pathological effects. However, it is very difficult to directly observe AChR internalization by AChR autoantibodies at the cellular level. Therefore, the present inventors have developed the AChR-5-HT3 of the present invention. A Using cell lines, we investigated whether AChR autoantibodies can induce AChR internalization. As a result, we confirmed that AChR autoantibodies can induce AChR internalization, and that the degree of AChR internalization varied depending on the type of autoantibody (Figures 7a and 7b).

[0094] Example 10: Measurement of cell surface expression

[0095] Chimeric autoantibody receptor (CAAR) is a technology that can be used to treat autoimmune diseases by specifically eliminating cells with BCR (B-cell receptor) for antibodies. The AChR-5-HT3 of the present invention A To determine whether the fusion protein can be used to develop immune cell therapy such as CAAR-T, AChR-5-HT3 was expressed in human T cells. A The expression was confirmed by introducing mRNA of AChRα1-5-HT3. A CAAR customs sequentially bound to genes, 4-1BB co-stimulatory domain, and CD3z, and AChRβ1-5-HT3 A , AChRδ-5-HT3 A , AChRε-5-HT3 AA total of four types of mRNA, including 1, were introduced into T cells via electroporation. Expression of AChR fusion proteins on the T cell surface was confirmed using fluorescently labeled AChR autoantibodies and bungarotoxin, and cell surface expression was detected in T cells introduced with fusion protein mRNA (Fig. 8).

[0096] Example 11: Measurement of fetal AChR cell surface expression

[0097] The present inventors analyzed whether autoantibody binding occurred due to fetal form AChR expression. Specifically, the prepared AChRα1-5-HT3 A , AChRβ1-5-HT3 A , AChRγ-5-HT3 A, and AChRδ-5-HT3 A Introduction of four genes into 293T cells (AChR-5-HT3) A ) and then measured AChR cell surface expression through flow cytometry using fluorescently labeled AChR-binding bungarotoxin and various types of AChR-binding autoantibodies (mAb35, mAb587, mAb192, and mAb637). As a result, it was confirmed that even when fetal AChR was expressed, it bound well to autoantibodies (Fig. 9), but when Alpha1 alone was expressed, surface expression by autoantibodies was not detected (Fig. 10).

[0098] In conclusion, the present inventors developed a fusion protein for diagnosing myasthenia gravis that can express a large amount of AChR autoantibody binding sites on the cell surface through the above study, and the fusion protein (cell line) can measure complement activation and receptor internalization by autoantibodies and measure the pathological action of AChR autoantibodies, so it can be used for detecting AChR autoantibodies appearing in myasthenia gravis patients, measuring pathological activity, and accurately diagnosing myasthenia gravis.

[0099] While the present invention has been described with reference to the above-described embodiments, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible. Therefore, the true scope of technical protection of the present invention should be determined by the technical spirit of the appended claims.

[0100] (National Research and Development Project that supported this invention)

[0101] This study, which is about the development of antibody treatment for myasthenia gravis, was conducted with the support of the National New Drug Development Program of the National Drug Development Fund of the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, and the Ministry of Health and Welfare (Project ID: RS-2023-00217215).

Claims

1. A fusion protein in which the extracellular domain of a nicotinic acetylcholine receptor unit is linked to the N-terminus of the transmembrane / cytosolic domain of a homotypic pentameric membrane protein excluding the acetylcholine receptor.

2. In paragraph 1, The above homomeric pentameric membrane protein is 5-HT3 A , 5-HT3 B , 5-HT3 C , 5-HT3 D and 5-HT3 E 5-HT3 receptors selected from the group consisting of β1, β1S265N, β2, β3, ρ1, ρ2 and ρ3; GABA selected from the group consisting of A A fusion protein selected from the group consisting of a receptor, an Erwinia chyrysanthemi ligand-gated ion channel (ELIC), a glycine receptor selected from the group consisting of α1, α2, α3 and α4, a Gloeobacter violaceus Ligand-Gated Ion Channel (GLIC), a formate-nitrite transporter (FNT) and a Mycobacterium tuberculosis mechanosensitive channel of large conductance (MscL).

3. In paragraph 2, The above 5-HT3 A A fusion protein, wherein the transmembrane / cytoplasmic domain comprises an amino acid sequence represented by SEQ ID NO:

12.

4. In paragraph 1, The above acetylcholine receptor unit is a fusion protein, wherein the AChR is a fetal form AChR selected from the group consisting of AChRα1, AChRβ1, AChRδ and AChRγ or an adult form AChR selected from the group consisting of AChRα1, AChRβ1, AChRδ and AChRε.

5. An artificial acetylcholine receptor produced by pentamerization of the fusion protein of clause 1 and having the ligand binding function of an acetylcholine receptor.

6. In paragraph 5, An artificial acetylcholine receptor having a ratio of AChRα1, AChRβ1, AChRδ, and AChRγ of 2:1:1:1 or a ratio of AChRα1, AChRβ1, AChRδ, and AChRε of 2:1:1:

1.

7. In paragraph 6, An artificial acetylcholine receptor, wherein the extracellular domain of the AChRα1 comprises an amino acid sequence represented by SEQ ID NO: 7, the extracellular domain of the AChRβ1 comprises an amino acid sequence represented by SEQ ID NO: 8, the extracellular domain of the AChRδ comprises an amino acid sequence represented by SEQ ID NO: 9, the extracellular domain of the AChRε comprises an amino acid sequence represented by SEQ ID NO: 10, and the extracellular domain of the AChRγ comprises an amino acid sequence represented by SEQ ID NO:

11.

8. A polynucleotide encoding the fusion protein of paragraph 1.

9. A recombinant vector comprising the polynucleotide of Article 8.

10. A transformed cell into which the recombinant vector of clause 9 has been introduced.

11. In paragraph 10, A transformed cell expressing an artificial acetylcholine receptor on the cell surface by introducing AChRα1, AChRβ1, AChRδ, and AChRγ in a ratio of 2:1:1:1 or by introducing AChRα1, AChRβ1, AChRδ, and AChRε in a ratio of 2:1:1:

1.

12. In paragraph 10, Transformed cells in which the gene encoding a complement inhibitor has been knocked out.

13. In paragraph 12, The above complement inhibitors are CD45, CD55 and CD59, transformed cells.

14. A composition for diagnosing myasthenia gravis, comprising the transformed cell of Article 10 as an active ingredient.

15. A step of treating the transformed cells of clause 10 with acetylcholine receptor-specific autoantibodies and the serum of the subject; A step of confirming the pathological activity of the transformed cells and acetylcholine receptor-specific autoantibodies; and A method for providing information for diagnosing and treating myasthenia gravis through detection of acetylcholine receptor-specific autoantibodies in the serum of a subject, comprising a step of confirming the severity of myasthenia gravis and determining a treatment method in a subject who exhibits similar pathological activity to a positive control group treated with human serum containing acetylcholine receptor autoantibodies and significantly higher pathological activity than a negative control group treated with normal serum.

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