JP2011173793A - Antibody for specifically recognizing aglycon having acetylcholine receptor cluster formation-inhibiting activity, acetylcholine receptor cluster formation ability-promoting agent containing the antibody, and aglycon-removing column filled with the antibody having acetylcholine receptor cluster formation ability-inhibiting activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop agents for treating diseases caused by the damage of acetylcholine receptor cluster formation ability. <P>SOLUTION: It has been newly found that the splicing variant of aglycon having a function for inhibiting acetylcholine receptor cluster formation ability exists. On the basis of the knowledge, it has been found that an antibody for specifically recognizing the aglycon having an acetylcholine receptor cluster formation-inhibiting activity can be used for the acetylcholine receptor cluster formation ability-promoting agent and an aglycon-removing column having acetylcholine receptor cluster formation-inhibiting activity. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an antibody specifically recognizing agrin having acetylcholine receptor cluster formation inhibitory activity, an acetylcholine receptor cluster formation promoter containing the antibody, and agrin having acetylcholine receptor cluster formation inhibitory activity filled with the antibody. A removal column.

The junction between motoneuron axon terminals and skeletal muscle cells is a highly differentiated anatomy called the neuromuscular junction (NMJ). When action potential reaches the nerve endings transmitted axon from the cell body of the motor neuron, voltage-dependent calcium nerve endings (Ca ²⁺⁾ channel (voltage-gated calcium channel; VGCC ) is opened Ca ²⁺ extracellularly Flows into the synaptic vesicle and acetylcholine (ACh) is released into the synaptic cleft. When ACh binds to the nicotinic acetylcholine receptor (nAChR) present in the endplate, the cation channel built into the receptor opens, generating an excitatory post-synaptic potential. This depolarizing membrane stimulation further activates voltage-gated Na ⁺ channels, spreading action potentials throughout the muscle. The action potential is detected by the dihydropyridine receptor present in the transverse tubule, and when the ryanodine receptor present in the sarcoplasmic reticulum membrane is activated, Ca ²⁺ in the sarcoplasmic reticulum is released into the cytoplasm. Increased intracellular Ca ²⁺ concentration initiates the interaction of muscle contraction-related proteins such as actin and myosin, causing muscle contraction. The NMJ is the key to this series of information transmission.

  The most characteristic phenomenon of NMJ differentiation is that nAChR assembles into the postsynaptic membrane during NMJ formation. This phenomenon is called cluster formation, and nAChR aggregates are called clusters. Various factors have been considered to be involved in the differentiation and formation of NMJ. In particular, post-synaptic membrane differentiation is thought to be important, and many experiments have been reported to support it.

  Myasthenia gravis (MG) is an autoimmune disease that targets proteins on the NMJ's postsynaptic membrane. The condition is inhibition of neuromuscular transmission due to synaptic dysfunction. The clinical feature is that patients complain of skeletal muscle fatigue and relieve after rest. Since 80% of MG patients are accompanied by thymic tumors and hyperplasia, thymic abnormalities are thought to be involved in the onset. MG depends on the type of autoantibody (1) anti-nAChR antibody-positive MG, (2) anti-muscle-specific tyrosine kinase (MuSK) antibody-positive MG, and (3) double seronegative in which the above antibody is not detected Classified as MG. About 80% of MG patients are equivalent to (1).

As the mechanism of action of the anti-nAChR antibody in the above (1), inhibition of the binding of ACh and nAChR, promotion of nAChR decay, complement-mediated post-synaptic membrane destruction, etc. have been considered. In particular, the decrease in nAChR number associated with complement-mediated post-synaptic membrane disruption is thought to be the essence of the disease state.
On the other hand, anti-nAChR antibody is not detected in (2) above, and unlike (1) above, most anti-MuSK antibodies belong to the IgG4 subclass and do not involve complement-mediated postsynaptic membrane disruption (Hoch, W., McConville, J., Helms, S., Newsom-Davis, J., Melms, A. and Vincent, A. (2001). Auto-antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor antibodies. Med. 7, 365-368.}. It is currently unclear how anti-MuSK antibodies cause MG symptoms.

  MuSK is a transmembrane phosphorylase with a molecular weight of 110 kDa and is adjacent to nAChR on the fascia. This MuSK is phosphorylated by agrin stimulation, and this phosphorylation promotes cluster formation of nAChR via rapsin located downstream. The agrin-MuSK-rapsin-nAChR signaling pathway that causes nAChR clustering has been speculated to be regulated at various levels, but has not yet been elucidated.

  In addition, the following patent documents relating to agrin or MuSK are disclosed.

  Patent Document 1 discloses that “the shortened delta 9 site of human agrin induces phosphorylation of MuSK receptor”.

  Patent Document 2 discloses “a method for treating a patient with congenital muscular dysfunction caused by LAMA2 gene mutation, which is characterized by administering an agrin-derived substance”.

  Patent Document 3 discloses “a method for diagnosing a neurotrypsin-related disease using an agrin fragment as a marker”.

  However, the above document does not disclose or suggest the relationship between the structure and function of an agrin molecule involved in acetylcholine receptor cluster formation.

Special Table 2000-504929 US70778379 Japanese Patent Laid-Open No. 5-304993

  An object of the present invention is to solve the development of a therapeutic agent for a disease caused by an impaired acetylcholine receptor clustering ability. More specifically, we elucidated the structural diversity and functions of agrin molecules involved in acetylcholine receptor cluster formation, and aimed to develop novel therapeutic agents for diseases caused by impaired acetylcholine receptor cluster formation ability.

In order to solve the above-mentioned problems, the present inventors performed identification of an agrin splicing variant by cDNA cloning and functional analysis of the expressed protein.
As a result of the functional analysis, it was newly found that there is a splicing variant of agrin having a function of inhibiting the ability to form an acetylcholine receptor cluster.
Based on the above findings, the present inventors have found that an antibody specifically recognizing agrin having acetylcholine receptor cluster formation inhibitory activity has an acetylcholine receptor cluster formation promoter and an acetylcholine receptor cluster formation inhibitory activity. It was found that it can be used for an agrin-removal column.

That is, the present invention is as follows.
1. An antibody that specifically recognizes SC2 type agrin and / or z0 type agrin.
2. The antibody according to item 1 above, wherein a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 is used as an immunogen.
Glu-His-Trp-Gln-Pro-Arg-Ala-Glu-Thr (SEQ ID NO: 1)
3. 3. The antibody according to item 1 or 2 having the following characteristics.
(1) deletion, substitution, insertion or addition of one to several amino acid residues in the amino acid sequence represented by Glu-His-Trp-Gln-Pro-Arg-Ala-Glu-Thr (SEQ ID NO: 1); Alternatively, a peptide having an amino acid sequence obtained by modification is used as an immunogen (2) Does not bind to SC1-type agrin, z8-type agrin, z11-type agrin and / or z19-type agrin. 4. The antibody according to any one of items 1 to 3, which is a monoclonal antibody.
5. An acetylcholine receptor cluster forming ability promoter comprising the antibody according to any one of items 1 to 4.
6). 6. The acetylcholine receptor cluster forming ability promoter according to 5 above, wherein the promoter is any one of the following therapeutic agents.
(1) Myasthenia gravis
(2) Amyotrophic lateral sclerosis
(3) Congenital muscular dystrophy (4) Alzheimer's disease (5) Parkinson's disease 5. An agrin removal column having an acetylcholine receptor cluster formation inhibitory activity, which is packed with the antibody according to any one of 1 to 4 above.
8). The column is used for the following therapeutic purposes.
(1) Myasthenia gravis
(2) Amyotrophic lateral sclerosis
(3) Congenital muscular dystrophy (4) Alzheimer's disease (5) Parkinson's disease A screening method for a substance that inhibits acetylcholine receptor cluster formation inhibitory activity of z0-type agrin or SC2-type agrin, comprising the following steps;
(1) a step of adding z0-type agrin and / or SC2-type agrin and a candidate inhibitor into cells,
(2) A step of measuring the inhibitory activity of a candidate inhibitory substance using the ability to form an acetylcholine receptor cluster in the cell as an index.

  The present inventors have made it possible to provide an antibody capable of specifically recognizing agrin based on the structure of agrin having an activity of inhibiting acetylcholine receptor cluster formation, which has not been conventionally known. Furthermore, the antibody can be used in an agrin removal column having an acetylcholine receptor cluster formation promoter and an acetylcholine receptor cluster formation inhibitory activity.

The result of amplifying the (C) terminal site of agrin for multiple samples by PCR. The arrow in the figure shows a typical z0-type agrin band having a normal C-terminal length. Comparison diagram of cDNA sequences of various agrin transcripts. The comparison figure of the C terminal amino acid sequence of various agrins. In the figure, the Y site (KSRK) is indicated by a double underline, the z8 insertion site (ELTNEIPA) is indicated by an underline, and the z11 insertion site (PETLDSRALFS) is indicated by a dotted underline. Each enclosed shows the agrin-specific amino acid sequence (e.g. amino acid sequence EHWQPRAET is the sequence of the specific SC2 (s hort C -terminal variant- 2 ) agrin). Schematic diagram of alternative splicing of various agrin mRNAs. Each box in the figure represents an exon. Schematic diagram showing the domain structure of various agrins {the topmost agrin is fluagrin (full-length agrin), and the others are miniagrins}. The schematic diagram which shows expression of various miniagrins. The figure of the result of the immunoblot analysis of various expressed miniagrin. Observation and evaluation results of nAChR cluster formation. The arrow in the figure indicates that an nAChR cluster is formed. Evaluation of nAChR cluster formation inhibitory activity of z0 type agrin and SC2 type agrin. The arrow in the figure indicates that an nAChR cluster is formed.

(Aglin)
Agrin is a type of heparan sulfate proteoglycan with a 225 kDa protein in the core, and has multiple putative glycosylation sites at the amino (N) terminal and central regions. Agrin is encoded by about 40 exons, and mRNA undergoes alternative splicing, resulting in different translation initiation points. For this reason, agrin with two types of N-terminals, short N-terminal (SN) consisting of 49 amino acids and long N-terminal (LN) consisting of 150 amino acids, is produced.
Furthermore, LN is divided into a secretory type with a signal sequence (SS) essential for secretion and an N-terminal agrin domain (NtA), which is a laminin binding region, and a transmembrane type with a transmembrane segment (TM). It is done.
On the other hand, there are three variable sites by alternative splicing of X, Y, and Z on the C-terminal side. Four amino acid insertions at the Y site are said to increase affinity with a-dystroglycan (a-DG). In addition to the z0 type without amino acid insertion, the Z site includes the z8, z11, and z19 types with 8, 11, and 19 amino acids inserted, and is collectively referred to as the z ⁺ type (Ferns, MJ, Campanelli , JT, Hoch, W., Scheller, RH, and Hall, Z. (1993). The ability of agrin to cluster AChRs depends on alternative splicing and on cell surface).

As is clear from Example 1 below, the present inventors presume that the z-, z8, and z19 types having the known standard type C-terminal encode a C-terminal shorter than the standard type. Two new variants were found. These SC1 (s hort C -terminal variant- 1 ), it was designated SC2 (s hort C -terminal variant- 2 ) agrin (see Figure 3).
Table 1 below shows the alternative splicing pattern revealed in past reports and in this example. The diversity of the agrin molecule is thought to have been created by multiplying the number of variable regions occurring at at least five locations at the N-terminus, X-site, Y-site, Z-site, and C-terminus.
Furthermore, the present inventors have newly found that SC2-type agrin and z0-type agrin have acetylcholine receptor cluster formation inhibitory activity, as is clear from Example 2 below.

(Antibodies specifically recognizing agrin with acetylcholine receptor cluster formation inhibitory activity)
The {antibody specifically recognizing agrin having acetylcholine receptor cluster formation inhibitory activity of the present invention (hereinafter sometimes referred to as "the antibody of the present invention")} refers to SC2 type agrin and / or z0 type agrin. It means an antibody that specifically recognizes, and is not particularly limited.
Furthermore, the antibody preferably does not bind to known z8 type agrin, z19 type agrin, and / or novel SC1 type agrin.
In addition, the antibodies include any type of polyclonal antibodies, antisera including polyclonal antibodies, or monoclonal antibodies, and fragments of these antibodies {Fab, F (ab ′) 2 or Fab ′ etc.} Is also included. Preferably, the antibody is a monoclonal antibody. Furthermore, the antibody is preferably converted into a humanized antibody by a method known per se.

{Immunogen (antigen) used to produce the antibody of the present invention}
As an immunogen for producing the antibody of the present invention, an amino acid sequence specific to SC2-type agrin and / or z0-type agrin derived from mammals, particularly preferably human or mouse, more specifically, z8-type agrin, z11-type A peptide containing all or part of an amino acid sequence that does not exist in agrin, z19-type agrin, and / or SC1-type agrin can be used.
As is clear from Example 1 below, SC2-type agrin has the following unique amino acid sequence (SEQ ID NO: 1: Reference FIG. 3) not found in other agrins. Therefore, preferably, a peptide comprising the following amino acid sequence can be used as an immunogen.
Glu-His-Trp-Gln-Pro-Arg-Ala-Glu-Thr (SEQ ID NO: 1)
Further, deletion, substitution, insertion or addition of one to several amino acid residues in the amino acid sequence represented by Glu-His-Trp-Gln-Pro-Arg-Ala-Glu-Thr (SEQ ID NO: 1), or A peptide comprising an amino acid sequence obtained by modification can also be used as an immunogen.

(Synthesis of immunogenic peptides)
Peptides can be produced by genetic engineering techniques, chemical synthesis, and cell-free protein synthesis. After the peptide is manufactured, it can be further purified and used.

  A peptide is produced by a general genetic engineering technique {edited by Sambrook et al., “Molecular Cloning, Laboratory Manual Second Edition”, 1989, based on the nucleotide sequence information or amino acid sequence of a gene encoding the peptide. , Cold Spring Harbor Laboratory; edited by Masaaki Muramatsu, “Lab Manual Genetic Engineering”, 1988, Maruzen Co., Ltd .; Ulmer, KM, “Science”, 1983, Vol. 219, p. 666- 671; edited by Ehrlich, HA, “PCR Technology, Principles and Applications of DNA Amplification”, 1989, Stockton Press}.

  Peptides can also be produced by general chemical synthesis methods. As a peptide chemical synthesis method, for example, a solid phase synthesis method, a liquid phase synthesis method, and the like are known, and any of them can be used.

  Peptide purification and / or separation can be performed by various separation operation methods utilizing its physical properties, chemical properties, and the like. Examples of the separation operation method include known methods such as ammonium sulfate precipitation, ultrafiltration, gel chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography, and dialysis. These methods can be used alone or in appropriate combination.

(Immune method)
A solution obtained by dissolving or suspending the purified peptide or partial peptide serving as the immunogen described above in an appropriate buffer such as a phosphate buffer (PBS) is used as the antigen solution. The antigen solution may be usually prepared to a concentration containing about 50 to 500 μg / mL of the antigen substance. In addition, when the antigen alone is low by the peptide alone, it can be used after being crosslinked with an appropriate carrier protein such as albumin or keyhole limpet hemocyanin (KLH).
Examples of animals (immunized animals) immunized with the antigen include mice, rats, hamsters, horses, goats, rabbits, and the like. Preferred are mice, more preferred are BALB / c mice.

  In order to enhance the responsiveness of the immunized animal to the antigen, the antigen solution can be mixed with an adjuvant and administered. Adjuvants that can be used here are Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi (MPL), Ribi (TDM), and Ribi (MPL + TDM). Examples include pertussis vaccine (Boredetella pertussis vaccine), muramyl dipeptide (MDP), aluminum adjuvant (ALUM), and combinations thereof, but combinations using FCA at the first immunization and FIA or Ribi adjuvant at the booster are particularly preferable. .

  The immunization method can appropriately change the injection site, schedule, etc. depending on the type of antigen to be used and the presence or absence of adjuvant mixing. (Antigen substance 10 to 200 μg) is injected intraperitoneally, subcutaneously, intramuscularly or (tail) intravenously, and boosted 1 to 4 times every about 4 to 21 days from the first immunization, and further about 1 to 4 weeks later Final immunization is performed. The antigen solution can also be administered without using an adjuvant by injecting intraperitoneally with an increased amount of antigen. The antibody titer is examined by collecting blood about 5 to 10 days after the booster immunization. The antibody titer can be measured by a conventional method according to the antibody titer assay described below. About 3 to 5 days after the final immunization, spleen cells are separated from the immunized animal to obtain antibody-producing cells.

(Production of monoclonal antibodies)
Monoclonal antibodies (hereinafter sometimes abbreviated as “MoAb”) are known per se, such as the method of Kohler and Milstein (Kohler G, Milstein C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497.).
For example, a hybridoma is prepared by recovering a tissue (eg, spleen or lymph node) containing antibody-producing cells from an immunized animal and fusing the antibody-producing cells with tumor cells (eg, bone tumor cells) of the antibody itself. Subsequently, after the hybridoma is cloned, the hybridoma producing the desired antibody is selected, and the antibody is recovered from the culture medium of this hybridoma.
As myeloma cells, those derived from mice, rats, humans, etc. are used, for example mouse myeloma P3X63-Ag8, P3X63-Ag8-U1, P3NS1-Ag4, SP2 / o-Ag14, P3X63-Ag8. Examples are myeloma cells. Some myeloma cells produce an immunoglobulin light chain, and if this is used as a fusion target, the immunoglobulin heavy chain produced by the antibody-producing cell and this light chain may bind randomly. In particular, it is preferable to use myeloma cells that do not produce an immunoglobulin light chain, such as P3X63-Ag8 · 653 and SP2 / o-Ag14.
The antibody-producing cells and the myeloma cells are preferably derived from the same species, particularly the same strain. The myeloma cells may be stored according to a method known per se. For example, those subcultured in a general medium supplemented with horse, rabbit or fetal calf serum are stored by freezing. For cell fusion, cells in the logarithmic growth phase are preferably used.

(Production of hybridoma)
Examples of a method for producing a hybridoma by fusing antibody-producing cells and myeloma cells include a method using polyethylene glycol (PEG), a method using Sendai virus, and a method using an electrofusion device. For example, in the case of the PEG method, splenocytes and myeloma cells are placed in an appropriate medium or buffer containing about 30 to 60% PEG (average molecular weight 1,000 to 6,000), 1 to 10: 1, preferably 5 to 10: 1. And the reaction may be carried out for about 30 seconds to 3 minutes under conditions of a temperature of about 25 to 37 ° C. and a pH of 6 to 8. After completion of the reaction, the cells are washed, the PEG solution is removed, the cells are resuspended in a medium, seeded in a microtiter plate, and culture is continued.
Cells after the fusion operation are cultured in a selective medium to select hybridomas. The selection medium is a medium in which the parent cell line can be killed and only the fused cells can grow, and a hypoxanthine-aminopterin-thymidine (HAT) medium is usually used. Selection of hybridomas is usually carried out by exchanging a part of the medium, preferably about half of the medium, with the selective medium 1 to 7 days after the fusion operation, and further culturing while repeating the same medium exchange every 2 or 3 days. The well where the hybridoma colony is growing is confirmed by microscopic observation.

In order to know whether the growing hybridoma is producing the desired antibody, the culture supernatant may be collected and an antibody titer assay may be performed by a method known per se.
Further, single clones are separated by a limiting dilution method, a soft agar method, or a method using a fluorescence excitation cell sorter.

  In order to examine the immunoglobulin subclass of the antibody produced by the hybridoma, the hybridoma is cultured under general conditions, and the antibody secreted in the culture supernatant is obtained using a commercially available antibody class / subclass determination kit or the like. You can know by analyzing.

(Method of obtaining monoclonal antibody)
The method for obtaining MoAb from the hybridoma can be appropriately selected depending on the required amount, the properties of the hybridoma, and the like. For example, a method of obtaining from mouse ascites transplanted with the hybridoma, a method of obtaining from the culture supernatant by cell culture, and the like are exemplified. A hybridoma capable of growing in the mouse abdominal cavity can obtain a high concentration of MoAb of several mg / mL from ascites. Hybridomas that cannot grow in vivo are obtained from the culture supernatant of the cell culture.
The acquisition of MoAb by cell culture has the advantage that the amount of antibody production is lower than in vivo, but there is little contamination with immunoglobulins and other contaminants contained in the mouse abdominal cavity, and purification is easy.
When the antibody is obtained from the abdominal cavity of a mouse transplanted with a hybridoma, for example, the intraperitoneal cavity of a BALB / c mouse previously administered with an immunosuppressive substance such as pristane (2, 6, 10, 14-tetramethylpentadecane). Hepaticoma (about 10 ⁶ or more) is transplanted, and ascites collected after about 1 to 3 weeks is collected. In the case of heterologous hybridomas (for example, mice and rats), it is preferable to use nude mice or radiation-treated mice.
When obtaining an antibody from the cell culture supernatant, for example, in addition to the stationary culture method used for cell maintenance, the hybridoma is cultured using a culture method such as a high-density culture method or a spinner flask culture method, and the antibody is contained. A culture supernatant is obtained.
Purification of MoAb from ascites or culture supernatant can be performed by a method known per se. For example, conventionally known fractionation methods by salting out using ammonium sulfate or sodium sulfate, polyethylene glycol fractionation method, ethanol fractionation method, DEAE ion exchange chromatography method, gel filtration method, etc. are applied as methods for purifying immunoglobulin. This is easily achieved.
Furthermore, when the MoAb is mouse IgG, it can be purified by affinity chromatography using a protein A-binding simple substance or an anti-mouse immunoglobulin binding simple substance, which is convenient.

{Screening method for substances that inhibit acetylcholine receptor cluster formation inhibitory activity of z0 type agrin or SC2 type agrin}
The screening method of the present invention includes at least the following steps.
(1) Step of adding z0-type agrin and / or SC2-type agrin and a candidate inhibitor to cultured cells The cell is not particularly limited as long as it can measure the ability to form an acetylcholine receptor cluster in the cell.
(2) A step of measuring the inhibitory activity of a candidate inhibitor using the ability to form an acetylcholine receptor cluster in the cell as an index. Acetylcholine receptor cluster in a cell to which a candidate inhibitor and z0-type agrin and / or SC2-type agrin are added. If the ability to form is high compared to the ability to form acetylcholine receptor clusters in cells supplemented with z0-type agrin and / or SC2-type agrin, the candidate inhibitor is acetylcholine of z0-type agrin or SC2-type agrin. It is a substance that inhibits receptor cluster formation ability inhibitory activity, in other words, a substance that can promote acetylcholine receptor cluster formation (acetylcholine receptor cluster formation promoting substance).
In addition, the following Example 2 can be referred for the measuring method of acetylcholine receptor cluster formation ability.

(Candidate inhibitor)
Any substance can be used as a candidate inhibitor used in the above screening. The type of candidate inhibitor is not particularly limited, and may be an individual small molecule synthetic compound, particularly siRNA, a compound present in a natural product extract, or a synthetic peptide. Alternatively, the candidate inhibitor can also be a compound library, a phage display library, or a combinatorial library. The candidate inhibitor is preferably a low molecular compound, and a compound library of low molecular compounds is preferred. The construction of a compound library is known to those skilled in the art, and a commercially available compound library can also be used.

(Acetylcholine receptor cluster formation promoter)
The “acetylcholine receptor cluster forming ability promoter” of the present invention refers to inhibiting the action of SC2 type agrin and / or z0 type agrin having a function of suppressing, inhibiting and / or reducing acetylcholine receptor cluster formation, or these agrins. And promotes the formation of acetylcholine receptor clusters.
The characteristics of the promoter of the present invention are clearly different from those of conventional drugs or acetylcholine degrading enzyme inhibitors that increase acetylcholine production. Conventional drugs that increase acetylcholine output can increase acetylcholine output in the patient's body or inhibit acetylcholine degradation, but if there are few acetylcholine receptors (if clusters are not formed) Acetylcholine could not bind to the acetylcholine receptor (nicotinic acetylcholine receptor), the cation channel built in the receptor could not be opened, and no excitatory post-synaptic potential was generated.
However, since the promoter of the present invention has a mechanism different from that of the conventional drug and promotes the ability to form an acetylcholine receptor cluster, even in patients with a small amount of acetylcholine production, a small amount of acetylcholine is efficiently converted into an acetylcholine receptor. The cation channel built into the receptor can open and generate an excitatory post-synaptic potential.
Furthermore, the promoter of the present invention is considered to be useful for a patient who has an excellent effect, in particular, a patient who is not effective with a conventional drug, by combining or combining with a conventional drug that increases acetylcholine output or an acetylcholine degrading enzyme inhibitor. It is done.

The promoter of the present invention comprises, as an active ingredient, an antibody that specifically recognizes agrin having acetylcholine receptor cluster formation inhibitory activity described above and / or an acetylcholine receptor cluster formation promoter obtained by the screening method described above. Including.
Furthermore, depending on the purpose of treatment (including prevention), various forms such as powders, granules, tablets, capsules, intestinal solvents, liquids, injections (liquids, suspensions) or forms used for gene therapy In addition, it can be prepared according to a conventional method.
In addition, fillers, extenders, binders, wetting agents, disintegrants, lubricants, diluents and excipients can also be included. In addition, stabilizers, bactericides, buffers, isotonic agents, chelating agents, surfactants, pH adjusters, and the like can be used as appropriate.

  The dosage or intake of the promoter of the present invention is not particularly limited as long as the effects of the present invention can be obtained, and the effectiveness of the components contained, the dosage form, the administration route, and the type of disease Depending on the nature of the subject (weight, age, medical condition, presence or absence of use of other medicines, etc.) and the judgment of the doctor in charge, etc. are appropriately selected. The promoter of the present invention can be administered or ingested in 1 to several times a day, and may be intermittently administered or ingested once every several days or weeks.

(Use of the accelerator of the present invention)
The promoter of the present invention can be used as a therapeutic agent for diseases associated with a decrease in acetylcholine and a decrease in acetylcholine receptor cluster-forming ability, for example, the following diseases.
(1) Myasthenia gravis
(2) Amyotrophic lateral sclerosis
(3) Congenital muscular dystrophy (4) Alzheimer's disease (5) Parkinson's disease

(Agrin removal column with acetylcholine receptor cluster formation inhibitory activity)
The “agrine removal column having acetylcholine receptor cluster formation inhibitory activity” of the present invention is packed with an antibody that specifically recognizes agrin having acetylcholine receptor cluster formation inhibitory activity described above. More specifically, the column is packed with an antibody that specifically recognizes SC2 type agrin and / or z0 type agrin.
The carrier for adsorbing the antibody is not particularly limited, and organic or inorganic porous materials such as cellulose gel, dextran gel, agarose gel, polyacrylamide gel, porous glass, and vinyl polymer gel can be used. A carrier used in ordinary affinity chromatography can be used.
In addition, the method for binding and / or adsorbing the antibody to a carrier can be performed by a known method such as a physical adsorption method, a chemical binding method or a combination thereof known per se.
Furthermore, if necessary, a molecule (spacer) of any length can be introduced between the carrier and the antibody.
In order to suppress non-specific reactions, etc., by a known method such as bovine serum albumin, surfactant, skim milk powder or the like is coated on the surface or inner wall of the carrier on which the antibody is immobilized. You may process and perform a blocking process.

When the column of the present invention is used as an extracorporeal circulation column, there are roughly the following two methods. For example, after separating blood taken from the body into a plasma component and a blood cell component using a centrifuge or a membrane plasma separator, the plasma component is passed through the column, and SC2 type agrin and / or Alternatively, after removing z0-type agrin and returning it to the body together with the blood cell component, the other is to pass the blood taken from the body directly through the column, and to remove SC2 type agrin and / or z0 type agrin. It is a method of removing.
One method for treating myasthenia gravis is a therapy that removes anti-acetylcholine receptor antibodies in plasma by simple plasma exchange therapy. The therapy using the column of the present invention can be performed with reference to the simple plasma exchange therapy for myasthenia gravis.

(Use of the column of the present invention)
It is considered that the column of the present invention can be used for the treatment of diseases involving reduction of acetylcholine and a decrease in the ability to form acetylcholine receptor clusters, for example, the following diseases.
(1) Myasthenia gravis
(2) Amyotrophic lateral sclerosis
(3) Congenital muscular dystrophy (4) Alzheimer's disease (5) Parkinson's disease

{Myasthenia gravis (MG)}
In MG, complement-mediated post-synaptic membrane disruption caused by anti-nAChR antibodies and concomitant decrease in nAChR number cause the pathophysiology, which causes neuromuscular transmission impairment.
That is, by administering the promoter of the present invention to an MG patient or removing SC2 type agrin and / or z0 type agrin from an MG patient by the column of the present invention, acetylcholine is efficiently produced even with a small expression amount of nAChR. Since it can bind to nAChR, it is considered to have a therapeutic effect on myasthenia gravis.

{Amyotrophic lateral sclerosis (ALS)}
Amyotrophic lateral sclerosis is one of the progressive neurodegenerative diseases characterized by motor neuron degeneration and atrophy and skeletal muscle paralysis. In addition, it has been reported that SOD mice, which are ALS model mice, show a decrease in agrin expression at the end of the pathological state (Dobrowolny, G., Giacinti, C., Pelosi, L., Nicoletti, C., Winn, N., Barberi, L., Molinaro, M., Rosenthal, N., and Musaro, A. (2005). Muscle expression of a local Igf-1 isoform protects motor neurons in an ALS mouse model. Biol. 168, 193-199).
That is, by administering the promoter of the present invention to an ALS patient or removing SC2 type agrin and / or z0 type agrin from an ALS patient using the column of the present invention, acetylcholine is efficiently produced even with a small amount of agrin expressed. Since it can bind to nAChR, it is considered to have a therapeutic effect on amyotrophic lateral sclerosis.

{Congenital muscular dystrophy (CMD)}
Congenital muscular dystrophy is a general term for muscular dystrophy that develops in the early childhood due to muscle weakness, joint contracture, and motor developmental delay. It is roughly classified into non-FCMD, which does not show CNS symptoms. Some non-FCMDs are caused by mutations in the gene LAMA2, which directs the α2 chain of laminin expressed in muscle fibers, and CMD model mice exhibit symptoms similar to those of human laminin-2 deficient congenital muscular dystrophy. Degeneration of muscle fibers in this disease fails to form the primary laminin skeleton required for the basement membrane structure, and the binding of the muscle basement membrane to the dystrophin-glycoprotein complex (DGC) or muscle This is thought to be because the binding between the basement membrane and integrins is lost. When intraperitoneal injection of adeno-associated virus incorporating z-type mini-agrin cDNA into this CMD model mouse, mini-agrin binds to α-DG, which is a component of the basement membrane and DGC, and mediated by mini-agrin Reported that the pathogenesis of muscle was improved by the stabilization mechanism of α-DG and laminin α5 chain (Moll, J., Barzaghi, P., Lin, S., Bezakova, G., Lochmuller, H., Engvall, E., Muller, U., and Ruegg, MA (2001). An agrin minigene rescues dystrophic symptoms in a mouse model for congenital muscular dystrophy. Nature 413, 302-307.).
That is, by administering the promoter of the present invention to a CMD patient or removing SC2 type agrin and / or z0 type agrin from a CMD patient using the column of the present invention, acetylcholine is efficiently produced even with a small amount of agrin expression. Since it can bind to nAChR, it is considered to have a therapeutic effect on congenital muscular dystrophy.

  Alzheimer's disease is known to be caused by impaired central acetylcholine receptor transmission. Parkinson's disease is known to be caused by impaired central dopamine transmission. Therefore, by administering the promoter of the present invention to a patient with Alzheimer's disease or Parkinson's disease or removing SC2 type agrin and / or z0 type agrin from the patient's blood by the column of the present invention from the patient, the therapeutic effect is obtained. It is believed that there is.

  Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following examples.

(Clone cloning of agrin splicing variant and expression of its protein)
In this example, as an example of a mammal, various cultured cell lines derived from mouse neural tissue and neuromuscular tissue were used to identify agrin splicing variants by cDNA cloning and to perform protein expression experiments thereof. Details are as follows.

(1) Cell culture Mouse myoblast-derived cultured cell line C2C12 was purchased from the American Type Culture Collection (Manassas, VA, USA). Mouse motor neuron-derived cell line NSC-34 was provided by Dr. Takeshi Tahira (National Institute for Longevity Medicine). C2C12 cells, NSC-34 cells, and monkey kidney fibroblast-derived COS-7 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; GIBCO BRL, Grand Island, NY, USA) with 10% fetal bovine serum (fetal bovine serum; FBS; GIBCO BRL), penicillin 100 units / ml (GIBCO BRL) and streptomycin 100 μg / ml (GIBCO BRL) were used. During passage, the culture medium is removed by aspiration, and adherent cells are washed with Hank's balanced salt solution (HBSS; GIBCO BRL) containing Mg ²⁺ and Ca ²⁺ , 0.25% trypsin, 1 mM ethylenediamine Mg ²⁺ and Ca ²⁺ -free HBSS (GIBCO BRL) containing acetic acid were added and reacted for 10 minutes in a CO ₂ incubator (37 ° C., 5% CO ₂ ; BNA-111; TABAI ESPEC, Osaka).
The detached cells were collected and centrifuged at 1500 rpm and 20 ° C. for 5 minutes. After removing the supernatant by aspiration, it is suspended in a new culture solution, and the number of cells is adjusted to 5 × 10 ⁴ cells / ml. 5 ml of the cell is inoculated into a culture flask, and a CO ₂ incubator (37 ° C., 5% CO 2). _It was cultured in ₂ ). The culture medium was changed every 3 days and subcultured in 1 week.
Mouse neuroblastoma x mouse fibroblast hybrid cell line N1E-115 cells (Amano et al., 1972), final concentration of 5% fetal calf serum (FCS; GIBCO BRL), 1 mM in DMEM Subculture was performed using a culture solution added so as to be hypoxanthine, 0.16 mM d-thymidine, and 0.01 mM aminopterin.
In addition to the above culture, differentiation induction of C2C12 cells was also performed. On day 2 after passage, the culture medium was removed by aspiration and replaced with DMEM containing 2% horse serum (HS; GIBCO BRL) prepared for differentiation induction. The cells were further cultured for 4 days in a CO ₂ incubator (37 ° C, 5% CO ₂ ).

(2) Extraction of total RNA Adult mice (C57BL / 6, BALB / c) were deeply anesthetized by intraperitoneal injection of Nembutal. After dislocation of the cervical vertebrae, the carotid artery was cut with scissors to remove blood. The excised brain and spinal cord were washed with Mg ²⁺ , Ca ²⁺ -free phosphate buffered saline (phosphate-buffered saline (−); PBS (−)}), and 600 μl of Lysis Solution was added to 30 mg of tissue pieces. And a tissue suspension was obtained using a Teflon homogenizer. NSC-34 and N1E-115 cells are seeded at 3 × 10 ⁴ cells / ml in a 60 mm culture dish (IWAKI) and cultured for 4 days in a CO ₂ incubator (37 ° C, 5% CO ₂ ). The cells were collected by washing with PBS (−). Undifferentiated C2C12 cells were cultured in a 60 mm culture dish at 1 × 10 ⁴ cells / ml for 3 days, and the cells were collected in the same manner. Differentiated C2C12 cells were seeded in the same manner as undifferentiated cells, and collected after normal culture for 3 days and differentiation induction for 4 days. All cells were lysed by adding 350 μl of Lysis Solution.
Total RNA was extracted from the above tissue suspension and cell lysate using total RNA extraction / purification Kit Mini (Agilent Technologies CA, USA) as follows.
Lysis Solution 350 μl added and lysed tissue suspension and total volume of cell lysate are centrifuged by Mini Prefiltration (16000 × g, 3 min, 20 ° C), and 350 μl of supernatant is diluted with 70% ethanol (Amresco, OH, USA) mixed with 350 μl. Centrifuge 700 µl of ethanol / Lysis Solution mixture in a Mini Isolation Column (16000 × g, 30 seconds, 20 ° C), discard the filtrate, and centrifuge with Wash Solution 500 µl (16000 × g, 30 seconds, 20 The column was washed twice and centrifuged (16000 × g, 2 minutes, 20 ° C.). Distilled water (DW) 30 μl was added and allowed to stand for 1 minute. The RNA was eluted by centrifugation at 16000 xg for 1 minute at 20 ° C.
In order to remove DNA mixed in the obtained sample, DNA degradation treatment was performed. The total amount of the reaction solution was 50 μl, and each total RNA sample 0.4-1 μg, 40 mM Tris-HCl (pH 7.5), 8 mM MgCl ₂ , 5 mM dithiothreitol, DNase I 2.5 unit was contained. After reaction at 37 ° C. for 10 minutes, phenol / chloroform treatment, diethyl ether treatment and ethanol precipitation were carried out. RNA was finally dissolved in 11 μl of DW, and the concentration was measured using a UV spectrophotometer (Nano Drop Technologies).

(3) Reverse transcriptase-polymerase chain reaction
Transcriptor First Strand cDNA Synthesis Kit (Roche) from total RNA extracted from spinal cord of C57BL / 6 mice, whole brain of BALB / c mice, N1E-115 cells, NSC-34 cells, undifferentiated C2C12 cells and differentiated C2C12 cells Complementary DNA (cDNA) was synthesized using Applied Science, Mannheim, Germany.
The total volume of the reaction solution is 20 μl, each total RNA 0.4-1 μg, 50 mM Tris-HCl (pH 8.5), 30 mM KCl, 8 mM MgCl ₂ , 60 μM Random Hexamer Primer, RNase Inhibitor 20 unit, 10 mM dNTPs, AMV (Avian myeloblastosis virus) reverse transcriptase (10 units) was mixed, reacted at 25 ° C. for 10 minutes and further at 50 ° C. for 1 hour, added with 180 μl of DW and stored at −80 ° C. until use.
Four types of primers (AgAFw, AgBFw, AgCRv, and AgDRv; see Table 2) were synthesized based on the base sequence of the mouse agrin gene. Using each prepared cDNA as a template, PCR was performed with a reaction solution containing 1 × attached buffer, 0.4 mM dNTPs, 0.3 μM AgAFw primer, 0.3 μM AgDRv primer, 1 μl cDNA sample, and 1.0 unit of KOD FX DNA polymerase. After heat denaturation at 94 ° C. for 2 minutes, the reaction cycle of heat denaturation (98 ° C., 10 seconds), annealing (60.4 ° C., 30 seconds), and extension (68 ° C., 1 minute) was repeated 20 times. Furthermore, 1 μl of the obtained reaction product was again added to the reaction solution having the same composition, and the same reaction was performed for 25 cycles. The obtained PCR product was separated by electrophoresis on a 1% agarose gel, stained with ethidium bromide solution (1 μg / ml), and then observed and recorded using a UV sampler (FAS-III; TOYOBO, Osaka). did.

(4) Isolation of cDNA clone The RT-PCR reaction product of (3) above was electrophoresed, and the 0.9-1.1 kb DNA fragment was gelled using GENECLEAN II Kit (MP Biomedicals LLC, Irvine, CA, USA). Recovered from. The purified DNA fragment was prepared in a solution containing rTaq (TaKaRa, Shiga) {10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ , 0.25 mM dNTPs, 0.25 μM A1 AgBFw, 0.25 μM AgCRv primer, rTaq 0.25 unit, cDNA sample 30 μl} was reacted at 72 ° C. for 10 minutes, and A addition reaction was performed. From this reaction solution, a DNA fragment was purified using Min Elute PCR Purification Kit (QIAGEN) and dissolved in 10 μl of DW. 1 μl of the recovered DNA fragment was mixed with 0.5 μl of Salt Solution (Invitrogen, Carlsbad, CA, USA) and 0.5 μl of pCR 2.1-TOPO (Invitrogen), followed by ligation reaction at room temperature for 30 minutes and then at 4 ° C. for 6 hours. . Further, 25 μl of E. coli TOP10 strain competent cells were added to each reaction solution and reacted on ice for 30 minutes and subsequently at 42 ° C. for 30 seconds. Then, 250 μl of SOC medium was added and cultured with shaking at 37 ° C. for 1 hour.
Pour 60 μl of this solution on LB agar medium containing ampicillin (50 μl / ml) coated with 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal; 8 mg / ml). The cells were cultured at 37 ° C for 12 hours.
Next, white colonies were selected from the appearing colonies, and the plasmid was extracted as follows in order to confirm that the cDNA was integrated. White colonies were placed in a tube containing 5 ml of LB liquid medium containing ampicillin (50 μl / ml) and cultured with shaking at 37 ° C. and 200 rpm. After 12 hours, the culture was collected by centrifugation at 3000 rpm for 10 minutes, and the resulting cells were suspended in 250 μl of Buffer P1 containing RNase I (10 mg / ml) and transferred to another tube. Thereto, 250 μl of Buffer P2 was added and mixed by inversion. Immediately after that, 350 μl of Buffer N3 was added and mixed again by inversion, followed by centrifugation at 13000 rpm for 10 minutes. The obtained supernatant was transferred to a QIAprep spin column and centrifuged at 13000 rpm for 1 minute to remove the filtrate. After this, add 500 μl Buffer PB, centrifuge at 13000 rpm for 1 minute, discard the filtrate, add 750 μl Buffer PE, centrifuge at 13000 rpm for 1 minute, discard the filtrate, and further centrifuge at 13000 rpm for 1 minute to remove the remaining liquid. Excluded. The QIAprep spin column was transferred to another tube, 50 μl of DW was added, allowed to stand at room temperature for 1 minute, and a plasmid solution was obtained by centrifugation at 13000 rpm for 1 minute. DNA concentration was measured with a UV spectrophotometer (Nano Drop Technologies).

(5) Base sequence determination 300 ng of each extracted plasmid DNA was attached to the ABI PRISM BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) 1 x Big Dye ver1.1 Reaction Mix, 1 x Big This was performed by the dideoxy method using Dye Sequence Buffer, 0.8 μM synthetic primer. The reaction product was purified by BigDye XTerminator ^™ purification kit (Applied Biosystems), and the base sequence was determined using Sequence Scanner v1.0 and DNA Sequencing Analysis software Version 5.1 using ABI PRISM 3130 Genetic Analyzer (Applied Biosystems).

(6) Database analysis We accessed the homepage of the DNA Data Bank of Japan (DDBJ) and used BLAST version 2.2.18.

(7) Preparation of plasmid for protein expression (7-1) Preparation of partial fragment of mini-agrin cDNA
Heat denaturation: 98 ° C, 10 seconds, annealing: 62.2 ° C, 30 seconds, extension: 68 ° C using 1 µl of cDNA derived from N1E-115 cells as a template and KOD FX DNA polymerase (TOYOBO) and A1 AgEFw and AgGRv primers, respectively. , 35 cycles under 1 minute conditions, heat denaturation with B1 AgHFw and AgKRv primers: 98 ° C, 10 seconds, annealing: 54.4 ° C, 30 seconds, extension: 68 ° C, 35 cycles under 1 minute conditions, C1 A1 fragment 743 bp, B1 fragment amplified by 35 cycles of PCR using AgBFw and AgNRv primers, heat denaturation: 98 ° C, 10 seconds, annealing: 57.3 ° C, 30 seconds, extension: 68 ° C, 1 minute The 1275 bp, C1 fragment 879 bp was purified using the Min Elute PCR Purification Kit (QIAGEN, Hilden, Germany).
Solution prepared using rTaq (TaKaRa) for this purified PCR product {10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ , 0.25 mM dNTPs, each 0.25 μM specific primer, rTaq 0.25 unit, cDNA sample 30 μl}} was reacted at 72 ° C. for 10 minutes, and each reaction product was purified again using Min Elute PCR Purification Kit (QIAGEN) and dissolved in 10 μl of DW. In the present invention, DNA fragments obtained from each combination of AgEFw, AgGRv primer, AgHFw, AgKRv primer, AgBFw, and AgNRv primer were designated as A1, B1, and C1.
1 μl of each of the purified A1, B1, and C1 DNA fragments was mixed with 0.5 μl of Salt Solution (Invitrogen) and 0.5 μl of pCR 2.1-TOPO (Invitrogen), and ligation reaction was performed at room temperature for 30 minutes and at 4 ° C. for 6 hours. To each ligation reaction product, 25 μl of E. coli TOP10 strain competent cell was added and allowed to react on ice for 30 minutes. Thereafter, the mixture was reacted at 42 ° C. for 30 seconds, 250 μl of SOC medium was added, and cultured with shaking at 37 ° C. for 1 hour. X-gal (8 mg / ml) was applied to Luria-Bertani (LB) agar medium containing ampicillin (50 μl / ml) with a sterilized congeal rod and dried in an incubator. 60 μl each was plated on LB agar medium containing ampicillin (50 μl / ml) and cultured at 37 ° C. for 12 hours. Plasmid DNA was prepared in the same manner as (4) above (A2, B2, C2).

(7-2) Preparation of vector
pBluescript II KS (+) (pBSII KS (+); Stratagene) was digested with EcoRV and XhoI, XhoI and BamHI, or XbaI and SacI for 2 hours at 37 ° C. Treat the cleaved plasmid with bacterial alkaline phosphatase (BAP) at 37 ° C for 1 hour, migrate on a 1% agarose gel, adsorb on DEAE paper, collect it, and treat with phenol / chloroform and diethyl ether After ethanol precipitation, it was dissolved in 50 μl of DW and used as a vector.
pCR2.1-TOPO (Invitrogen) was digested with XbaI and SacI for 2 hours at 37 ° C. The digested plasmid was treated with BAP at 37 ° C. for 1 hour and electrophoresed on a 1% agarose gel. It was adsorbed on DEAE paper and collected. After phenol / chloroform treatment, diethyl ether treatment and ethanol precipitation, it was dissolved in 50 μl of DW and used as a vector.
pcDNA3.1 (+) (Invitrogen) was digested with EcoRV and NotI for 2 hours at 37 ° C., and the cleaved plasmid was treated with BAP at 37 ° C. for 1 hour and electrophoresed on a 1% agarose gel. It was adsorbed on DEAE paper and collected, and after phenol / chloroform treatment, diethyl ether treatment and ethanol precipitation, it was dissolved in 50 μl of DW and used as a vector.
Mammalian cell expression plasmid pEF-BOS (provided by Professor Shigekazu Nagata, Graduate School of Medicine, Kyoto University) was digested with XbaI at 37 ° C. for 1 hour and then BAP-treated at 37 ° C. for 1 hour. After phenol / chloroform treatment, diethyl ether treatment and ethanol precipitation, the product was dissolved in 50 μl of DW. In addition to 20 μl, add 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl ₂ , 1 mM DTT, 250 μM dNTPs, 0.01 units / μl Klenow fragment of DNA polymerase I The reaction was carried out at 20 ° C. for 20 minutes. After electrophoresis on a 1% agarose gel, it was recovered using GENECLEAN II Kit (MP Biomedicals LLC) and used as a vector.

(7-3) Construction of mini agrin cDNA
React each plasmid DNA of A2, B2, and C2 at 100 ° C for 10 minutes, 1 μl of which is used as a template, A2 plasmid is T7 and AgGRv primer, B2 plasmid is AgHFw and M13rev primer or T7 and AgKRv primer, C2 plasmid Was amplified with AgBFw and M13rev primer at an annealing temperature of 52.2 ° C. under 20 cycles using KOD FX polymerase (TOYOBO), purified using Min Elute PCR Purification Kit (QIAGEN), and dissolved in 15.4 μl of DW. The obtained DNA fragments were designated as A3, B3, B3 ′, and C3 in this order.
Terminal phosphorylation of A3, B3, B3 ′, C3 was performed at 37 ° C. for 30 minutes in a reaction solution containing 1 × Protruding End Kinase Buffer, T4 polynucleotide kinase 0.5-2 units / μl, 1 mM ATP, It was stopped by heating for 5 minutes. The DNA fragments obtained here were designated as A4, B4, B4 ′ and C4. Mix A4 1 μl and B4 1 μl, B4 ′ 1 μl and C4 1 μl, add Mighty mix 2 μl (TaKaRa) to each, perform ligation reaction at 16 ° C for 30 minutes, and mix reaction products with D1, E1 It was.
Further, PCR was performed with T7 and AgJRv primers using D1 as a template and T7 and M13rev primers using E1 as a template. KOD FX polymerase (TOYOBO) was used under conditions of an annealing temperature of 52 ° C. and 35 cycles. The reaction solution was electrophoresed on a 1% agarose gel, and a band near 1741 bp derived from D1 and a band near 2344 bp derived from E1 were collected using GENECLEAN II Kit (MP Biomedicals LLC), and were designated as D2 and E2, respectively. . D2 is digested with restriction EcoRV and XhoI, and E2 is digested with XhoI and BamHI, respectively. After electrophoresis on 1% agarose gel, fragments near 1153 bp from D2 and fragments near 1781 bp from E2 are recovered with GENECLEAN II Kit. D3 and E3.
1.5 μl of D3 fragment and 1.5 μl of E3 fragment were prepared by pBSII-KS (+) 1 μl (25 ng) cleaved with EcoRV and XhoI prepared in (7-2) above, pBS2-KS (+) cleaved with XhoI and BamHI ) 1 μl (25 ng) each, Mighty mix 2.5 μl (TaKaRa) was added to each, ligation was performed at 16 ° C. for 30 minutes, and the reaction products were D4 and E4, respectively. To this _{D4, E4 50 mM MgCl 2/} 10 mM CaCl 2 was added to 100 [mu] l E. coli SCS1 strain competent cell 100 [mu] l, and reacted on ice for 30 minutes.
Thereafter, the mixture was reacted at 42 ° C. for 1 minute, 250 μl of SOC medium was added, and reacted at 37 ° C. for 1 hour. 60 μl of this solution was plated on LB agar medium containing ampicillin (50 μl / ml) and cultured at 37 ° C. for 12 hours. Plasmids were extracted from the obtained colonies of D4 and E4 using Plasmid DNA purification QIAprep spin Miniprep Kit (QIAGEN), and these plasmids were designated as D5 and E5.
D5 was digested with XbaI and XhoI, E5 was digested with SacI and XhoI, and electrophoresed on a 1% agarose gel, and a fragment near 1181 bp derived from D5 and a fragment near 947 bp derived from E5 were generated by GENECLEAN II Kit (MP Biomedicals LLC ) To obtain D6 and E6, respectively. Mix 1 μl of D6 fragment, 1 μl of E6 fragment and XbaI prepared in (7-2) above and 1 μl (25 ng) of pCR2.1 vector cleaved with SacI, and then add Mighty mix 3 μl (TaKaRa). The ligation reaction was performed at 16 ° C. for 30 minutes, and the reaction product was designated as F1. Add 20 μl of E. coli DH5α competent cell (TaKaRa) to this F1 sample, react on ice for 30 minutes, react at 42 ° C for 45 seconds, add 200 μl of SOC medium, and add 1 μm at 37 ° C and 200 rpm. Cultured with shaking. 60 μl of this solution was plated on LB agar medium containing X-gal (8 mg / ml) -coated ampicillin (50 μl / ml) and cultured at 37 ° C. for 12 hours. The obtained F1 colonies were plasmid-extracted using Plasmid DNA purification QIAprep spin Miniprep Kit (QIAGEN), and this was designated as F2.
The F2 plasmid was digested with EcoRV and SacI, electrophoresed on a 1% agarose gel, and a fragment near 2092 bp was adsorbed on DEAE paper. This was eluted with 1.5 M NaCl / 10 mM Tris-HCl (pH 7.5), extracted with phenol / chloroform, treated with diethyl ether, precipitated with ethanol, and dissolved in 50 μl of DW. The obtained DNA fragment was designated as F3.
Each DNA fragment of the C-terminal variant was digested with SacI and NotI and electrophoresed on a 1% agarose gel, and then each band of 851-1108 bp was recovered with GENECLEAN II Kit (MP Biomedicals LLC). 1 μl of each fragment, 1 μl of F3 prepared in advance, and 1 μl (25 ng) of pcDNA 3.1 vector cleaved with EcoRV and NotI, add Mighty mix 3 μl (TaKaRa), mix and mix, A ligation reaction was performed at 16 ° C. for 30 minutes, and this reaction product was designated as G1. Add 20 μl of E. coli DH5α competent cell (TaKaRa) to this G1 sample, react on ice for 30 minutes, react for 45 seconds at 42 ° C, add 200 μl of SOC medium, and then at 37 ° C and 200 rpm for 1 hour. Cultured with shaking. 60 μl of this was plated on ampicillin (50 μl / ml) -containing LB agar medium coated with X-gal (8 mg / ml) and cultured at 37 ° C. for 12 hours. The obtained G1 colony was extracted with QIAprep spin Miniprep Kit (QIAGEN) to obtain an expression plasmid.
Furthermore, the cDNA incorporated into pcDA3.1 (+) was further excised with EcoRV and XhoI, the ends were blunted with Klenow fragment of DNA polymerase I, and incorporated into the pEF-BOS vector prepared in (7-2) above. And used as an expression plasmid.

(8) Gene transfer
COS-7 cells were prepared at 1 × 10 ⁵ cells / ml, 2 ml of which was seeded on a tissue culture 6-well plate (IWAKI) and maintained in a CO ₂ incubator (37 ° C., 5% CO ₂ ). One or two days after subculture, the culture medium was removed by suction and replaced with the same amount of DMEM-10% FBS. Mix 97 μl of DMEM and 3 μl of FuGENE 6 Transfection Reagent (Roche Applied Science), let stand for 5 minutes at room temperature, then mix 2 μg of expression plasmid incorporating various mini-agrin cDNAs described in (7-3) above. For 60 minutes. 100 μl of this mixture was added per well and cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ).

(9) Immunoblot analysis of expressed protein 24 hours after gene introduction, the culture medium was replaced with the same amount of FCS-free DMEM. After further culturing in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 24 hours, the supernatant was collected and centrifuged at 1500 rpm and 20 ° C. for 5 minutes. The obtained supernatant was further concentrated using Microcon YM-10 (Millipore, Billerica, MA, USA) to obtain an immunoblot sample. Equal volume of 2 × Sample Buffer (Tris Base 282 mM, Tris-HCl 212 mM, 146 nM lithium dodecyl sulfate, 2.18 M Glycerol, 1.02 mM EDTA, 100 mM DTT, 0.44 mM Serva Blue G250, 0.35 mM Phenol Red, pH 8.4; Invitrogen) and heated at 95 ° C. for 5 minutes. 5-15 μl of each sample (50 ng-3 μg) on 4-12% SDS polyacryl gel electrophoresis (NuPAGE 4-12% Bis-Tris Gel and NuPAGE MOPS SDS Running Buffer; Invitrogen) at 50 V at 200 V Electrophoresis was performed. The protein after electrophoresis was transferred to a PVDF membrane (Invitrogen) at 30 V over 60 minutes. The membrane after transfer was immersed in Tris-buffered saline (Tris-buffered saline; TBS; 20 mM Tris, 150 mM NaCl, pH 7.5) containing 3% BSA and blocked at room temperature for 1 hour. Subsequently, the membrane was washed 3 times for 10 minutes with TBS (TTBS) containing 0.05% Tween 20 (Bio-Rad Laboratories, CA, USA), and then a rabbit anti-agrin polyclonal antibody (sc-25528; SANTA CRUZ BIOTECHNOLOGY, Santa Cruz, CA, USA) was subjected to a primary antibody reaction using a 500-fold diluted solution of TTBS containing 1% BSA (1% BSA-TTBS). After reacting overnight at 4 ° C, the membrane was washed 3 times for 10 minutes each and then diluted 50,000 times with HRP-labeled goat anti-rabbit IgG antibody (MP Biomdicals, Inc.-Cappel Products, Costa Mesa, CA, USA) A secondary antibody reaction was performed with the solution. After reacting at room temperature for 1 hour, the membrane was washed three times for 10 minutes each and reacted with ECL Plus (GE Healthcare UK Ltd., Buckinghamshire, England) reagent for 5 minutes at room temperature. LAS-4000UVmini (Fujifilm, Tokyo) was used for signal detection. The detection method was set to chemiluminescence, and the exposure method was set to precision.

(Results of cDNA cloning of Agrin variant)
Total RNA was extracted from C57BL / 6 spinal cord, BALB / c whole brain, N1E-115 cells, NSC-34 cells, undifferentiated C2C12 cells, and differentiated C2C12 cells, and RT-PCR was performed. The primers were synthesized based on the known agrin mRNA translation region and the base sequence of the 3 ′ untranslated region. In the first set of primers (AgAFw and AgCRv; see: Table 2), non-specific amplification was observed at high frequency, so another set of primers (AgBFw and AgDRv; see: Table 2) was used and nested. PCR was performed. When the PCR product was electrophoresed on a 1% agarose gel, multiple bands in the range of 800-1200 bp were detected in all samples (FIG. 1).
In order to determine the base sequence of the amplified DNA fragment, the PCR product was collected from the agarose gel for each tissue and cell and incorporated into the pCR2.1-TOPO vector. E. coli competent cells were transformed, 6 white colonies obtained on an agar medium coated with X-gal were used as one pool, and plasmid DNA was extracted and PCR was performed. A pool that gave an amplification product longer than 916 bp expected from known z0-type agrin was selected, and individual colonies were similarly searched to obtain the desired cDNA clone. After this process, 9 C57BL / 6 mouse spinal cords, 7 BALB / c mouse whole brain, 6 N1E-115 cells, 2 NSC-34 cells, 2 undifferentiated C2C12 cells And 2 cDNA clones were obtained from differentiated C2C12 cells. As a result of determining the nucleotide sequence of the inserted cDNA and conducting a homology search using BLAST version 2.2.18, all the sequences were at least partially matched with known mouse agrin.

(Confirmation of diversity of agrin mRNA)
The PCR product derived from C57BL / 6 spinal cord contained z0 type without exon insertion at the Z site, z8 type with 24 bp exon insertion, and z19 type cDNA clone with 57 bp exon insertion. These nucleotide sequences have already been reported by Hoch et al. {Hoch, W., Ferns, M., Campanelli, JT, Hall, ZW, and Scheller, RH (1993) .Developmental regulation of highly active alternatively spliced forms of agrin. 11, 479-490.} Matched the splicing variant. In addition to these, we found two new variants. In one variant, so far the 35th intron has remained unspliced. In the case of this mRNA, since a different stop codon appears, the translation region was 102 bp shorter than the known z0 type (FIG. 4). The other variant had a stop codon due to the 30th intron remaining, and the C-terminal translation region was 543 bp shorter than the known z0 type. The protein products predicted from these mRNAs were estimated to be 86 amino acids and 233 amino acids shorter than the known C-terminal of agrin. SC2 in particular was expected to lack the entire LG3 domain (Figures 3 and 5). In the present invention, these types of SC1 (s hort C -terminal variant- 1 ), was designated SC2 (s hort C -terminal variant- 2 ) type. In addition, a z8-SC2 type cDNA clone having a 24 bp exon insertion at the Z site and having an intron remaining was isolated.
Moreover, from PCR products derived from BALB / c whole brain, there are z0 type without exon insertion at Z site, z19 type with 57 bp exon insertion, SC2 type with intron remaining, 57 bp exon insertion at Z site, And the z19-SC2 type with intron remaining was identified. From the PCR product derived from N1E-115 cells, z0 type without exon insertion at the Z site and SC2 type with intron remaining were identified. From the PCR product derived from NSC-34 cells, the z0 type without exon insertion at the Z site was identified. From the PCR product derived from undivided C2C12 cells, z0 type having no exon insertion at the Z site and z0-SC1 type having no exon insertion and intron remaining were identified. From the PCR product derived from differentiated C2C12, the z0 type without exon insertion at the Z site was identified (FIGS. 2 and 4).

(Protein expression result)
In order to investigate what kind of physiological function the splicing variant of the agrin molecule obtained this time is involved in, we established a protein expression system. Based on previous research reports, N-terminal is made to be LN type, and at the same time, 8 FS domains (follistatin-like domain) to which neuronal adhesion molecules and heparin-binding growth factor bind, 2 LE domains (laminin EGF) -like domain), one SEA domain (sperm protein enterokinase and agrin domain), and two S / T sites (serine / threonine sites) excluding the expression structure of the expression plasmid (Fig. 5) ). Three DNA fragments obtained by RT-PCR were combined from total RNA extracted from N1E-115 cells, and the N-terminal cDNA of mini-agrin as originally reported was incorporated into the expression plasmid pEF-BOS. Furthermore, the final expression plasmids of each variant were constructed by ligating the cDNAs of z0, z8, z19, SC1, and SC2 types on the C-terminal side (FIG. 6).
Next, the plasmid containing the mini-agrin cDNA was introduced into COS-7 cells, and the culture supernatant after 24-48 hours was collected to check whether secreted mini-agrin was produced or not by immunoblotting. Attempted detection. As a result, a single band was detected for the z0, z8, z19, and SC2 type samples (FIG. 7). The estimated molecular weights of z0, z8, z19, and SC2 type mini agrins are 109 kDa, 110 kDa, 111 kDa, and 84 kDa, respectively, and the molecular weights measured from each band in FIG. 7 are 113 kDa, 113 kDa, and 116 kDa. The value was close to 88 kDa.

(Evaluation of acetylcholine receptor cluster formation ability of each agrin)
The acetylcholine receptor cluster forming ability or inhibitory activity of each agrin expressed in Example 1 was confirmed. Details are as follows.

(Confirmation method of nACR cluster formation)
C2C12 cells were prepared at 5 × 10 ⁴ cells / ml, seeded at 2 ml per well of a 6-well plate for tissue culture (IWAKI), and cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ). After 24 hours, the medium was replaced with 2% HS-DMEM and cultured for 4 days to be differentiated. Recombinant agrin (Rat C-terminal Agrin; R & D SYSTEMS, Minneapolis, MN, USA) dissolved in 0.2% BSA-PBS (-) alone in wells where myotube was confirmed, secreted into the culture supernatant of COS-7 cells The various mini-agrins alone or a mixture of recombinant agrin (R & D SYSTEMS) and various mini-agrins secreted into the culture supernatant of COS-7 cells was added. As a negative control, each lysis dilution was added.
15 hours after the addition, the culture supernatant was removed, washed with PBS (−), 3.7% formaldehyde (16% ultrapure formaldehyde methanol free; Polysciences, Eppelheim, Germany) -PBS was added, and the cells were fixed at room temperature for 10 minutes. After washing with 1% BSA-PBS (-) three times for 1 minute each, fluorescently labeled a-bungarotoxin (a-bungarotoxin; a-BuTX, Alexa Fluor 488 conjugate; Molecular Probes, Eugene, OR, USA) Was added to a final concentration of 100 nM and allowed to react at room temperature for 1 hour in the dark. After washing twice with 1% BSA-PBS (−) for 5 minutes, Propong Gold antifade regent (Molecular Probes) was added and encapsulated. For the fluorescence observation, an inverted microscope (IX 71; OLYMPUS, Tokyo) equipped with a 20 × objective lens and a NIBA filter was used. A DP71 digital camera (OLYMPUS) and dedicated software (DP Controller and DP Manager; OLYMPUS) were used to capture the images. In the evaluation method, Alexa 488 signals with a diameter of 10 μm or more were counted as nAChR clusters. Five fields of view were taken for each well, the number of nAChR clusters per myotube was calculated for each field, and the average value per well was calculated. The value obtained by mock-trasfection was taken as 100%, and the ratio of each sample to that was converted to percentage. Significance test was performed by Tukey-Kramer test with a significance level of 1%.

(Evaluation of acetylcholine receptor cluster formation ability of each agrin)
The mini-agrine variant recovered from the COS-7 cell culture supernatant was added to the differentiation-induced C2C12 cells. At 15 hours after addition, visualization of nAChR was attempted using a-BuTX fluorescently labeled with Alexa 488. A cluster was defined as an aggregate of nAChR aggregated with a diameter of 10 μm or more. Cluster formation of nAChR was clearly observed in C2C12 cells to which recombinant agrin, z8 type, and z19 type miniagrin were added (FIG. 8A). The number of clusters per myotube is 835 ± 21% (n = 4), 911 ± 64% (n = 4), and 859 ± 103% (n = 4) when the mock-transfection value is 100%. Values are shown (FIG. 8B). In contrast, almost no cluster formation of nAChR was observed in C2C12 cells supplemented with z0 and SC2 type mini-agrins, similar to the supernatant of COS-7 cells transfected with vector only (mock-transfection) ( FIG. 8A). The number of clusters per myotube was 89 ± 21% (n = 4) and 106 ± 30% (n = 4), respectively, compared with mock-transfection (FIG. 8B).
Next, the cluster formation inhibitory activity of zA, SC2 type nAChR was examined. Compared to the value of 686 ± 77% (n = 4) with the addition of a mixture of mock-transfection and recombinant agrin, the mixture of z0 and recombinant agrin, and SC2 and recombinant agrin The mixtures showed values of 430 ± 24% (n = 4) and 498 ± 55% (n = 4), respectively, confirming a significant decrease in nAChR cluster formation (p <0.01; FIG. 9).

(General)
From the results of the above Examples, it was confirmed that z0 type agrin and SC2 type agrin have acetylcholine receptor cluster formation inhibitory activity. Thereby, it is considered that an antibody that specifically recognizes z0-type agrin and / or SC2-type agrin can inhibit the acetylcholine receptor cluster formation inhibitory activity of z0-type agrin and / or SC2-type agrin.

  The present inventors have made it possible to provide an antibody capable of specifically recognizing agrin having acetylcholine receptor cluster formation inhibitory activity. Furthermore, there is a possibility that the antibody can be used in an acetylcholine receptor cluster forming ability promoter and an agrin removal column that inhibits the acetylcholine receptor cluster forming ability.

Claims (9)

  An antibody that specifically recognizes SC2 type agrin and / or z0 type agrin. The antibody of Claim 1 which uses the peptide containing the amino acid sequence represented by following sequence number 1 as an immunogen.
Glu-His-Trp-Gln-Pro-Arg-Ala-Glu-Thr (SEQ ID NO: 1) The antibody according to claim 1 or 2, which has the following characteristics.
(1) deletion, substitution, insertion or addition of one to several amino acid residues in the amino acid sequence represented by Glu-His-Trp-Gln-Pro-Arg-Ala-Glu-Thr (SEQ ID NO: 1); Or, use a peptide consisting of an amino acid sequence obtained by modification as an immunogen (2) Does not bind to SC1-type agrin, z8-type agrin, z11-type agrin and / or z19-type agrin   The antibody according to any one of claims 1 to 3, which is a monoclonal antibody.   The acetylcholine receptor cluster formation ability promoter containing the antibody of any one of Claims 1-4. The acetylcholine receptor cluster formation ability promoter according to claim 5, wherein the promoter is any one of the following therapeutic agents.
(1) Myasthenia gravis
(2) Amyotrophic lateral sclerosis
(3) Congenital muscular dystrophy (4) Alzheimer's disease (5) Parkinson's disease   An agrin removal column having acetylcholine receptor cluster formation inhibitory activity, which is packed with the antibody according to any one of claims 1 to 4. The column is used for the following therapeutic purposes.
(1) Myasthenia gravis
(2) Amyotrophic lateral sclerosis
(3) Congenital muscular dystrophy (4) Alzheimer's disease (5) Parkinson's disease A screening method for a substance that inhibits acetylcholine receptor cluster formation inhibitory activity of z0-type agrin or SC2-type agrin, comprising the following steps;
(1) a step of adding z0-type agrin and / or SC2-type agrin and a candidate inhibitor into cells,
(2) A step of measuring the inhibitory activity of a candidate inhibitory substance using the ability to form an acetylcholine receptor cluster in the cell as an index.