TWI238251B - In-vitro adsorbing device using immunity - Google Patents

Abstract

The invention provides an in-vitro absorbing device using immunity that adsorbs only a specific substance. The in-vitro adsorbing device is based on a reaction between an antibody that is prepared by a genetic engineering and an antigen. Since the reaction is specific, a specific harmful substance in the patient's body can be practically eliminated.

Description

1238251 — __ Case No. 89113958 倏 倏 倏 五. V. Description of the invention (1) Field of the invention The present invention relates to an in vitro immunoadsorption device, in particular to the use of human acetylcholine receptors. Peptide, an in vitro immunoadsorption device that specifically adsorbs autoantibodies. BACKGROUND OF THE INVENTION Acetylcholine receptor (AChR) is the main receptor for neuromuscular conduction. Acetylcholine (ACh) released from nerve endings binds to AChR on muscle fibers and induces muscle cell membrane production.

The action potential causes muscle contraction. The main cause of myasthenia gravis (MG) is that the amount of AChR on muscle fibers is reduced or unable to perform functions, which affects the patient's muscle contraction. In severe cases, it affects respiratory muscles and causes death (861 ^ 〇 ¥, et al ·, 1992, 16th edition, Merck & Co., Inc. Rahway, NJ 1524-1526). The production of autoantibodies is found in 90% of patients' lysates. These antibodies are mainly recognizing tritium, with a concentration of 1 to 100 nM (Lindstrom et al., 1 976, Neurology, 26: 1054 ^ 1059; Lennon, 1994, Serological diagnosis of myasthenia gravis and the Lambert-Eaton

myasthenia syndromes. New York: Dekker, pp. 149-164), most of its antibody types are immunoglobulin * IgG. Autoantibodies not only cut off the role of AChR on the muscle membrane, but also destroy the AChR on the membrane, which is the main cause of such diseases caused by anti-AChR antibodies. In clinical practice, the treatment methods include: (1) Inhibiting AChR metabolism with drugs to increase the duration of ACh action (AquUionius, et ai.,

1238251 _ Case No. 89113958_ Year Month Date _ Amendment _ • Fifth, description of the invention (2) 1 983 J. Neurol. Neurosurg. Psych. 46: 929); (2) high dose of corticosteroids (cor ΐ icosteroid) Or immunosuppressants, reducing the production of autoantibodies (Pascuzzi et al., 1 984, Ann. Neurol, 43: 644-659; Goulon et al., 1988, Results of a one-year open trial of cyclosporin in ten patients with severe myasthenia gravis. In: Kahan, BD, ed. · Cyclosporin: applications in autoimmune disease.

Philadephia: Grune & Stratton, p. 211; Perez et. Al., 1981, Neurology 31: 32-38); (3) thymectomy; and (4) plasmapheresis to remove corporate fluid Autoantibodies. All in all, it is to reduce the autoantibodies in the blood circulation, Φ to achieve the purpose of treatment. Therefore, researchers have been developing extracorporeal systems to remove antibodies using the principle of immuno-adsorption. Existing devices include tryptophan-immobilized carriers (ImmusorbaTM) and protein A (sepharose). The former uses the principle of charge attraction (see U.S. Patent Nos. 4, 627, 91 5 and 4, 432, 871) to remove specific proteins but is not specific to antibodies; the latter works by the principle that protein A can bind IgG Fragment (Fc), so it can adsorb all IgG (see US Pat. No. 5, 753, 227), however, the autoantibodies that cause jig account for only one ten thousandth of the total I gG, with protein a As an adsorbent, not only the effect is limited, but the device valley is easily over-adsorbed and saturated, and it is necessary to perform another stripping step or even replace the device, which is quite disadvantageous in terms of cost. So ’still needs a device that specifically adsorbs autoantibodies,

0643-5413TWF3; s u s anwu.p t c Page 5 1238251 Amendment, No. 89113958 V. Description of the Invention (3) Treatment of autoimmune diseases politically, and can greatly reduce costs. Summary of the Invention In view of this, the present invention uses the AChR recognized by autoantibodies to express its genetic engineering method 'in addition to mass production, it is specific to autoantibodies' and will not affect other protein components in plasma. For mg, metallurgical therapy can have a direct effect.

... Therefore, one aspect of the present invention is to provide an in vitro immunoadsorption device for specifically adsorbing a specific substance, including: (a) a substrate; and (), a conjugate specifically adsorbed to the specific substance, which is connected to The matrix f may be filled in the matrix; wherein the conjugate includes a peptide, a fragment of a polypeptide f protein, an epitope, an antibody or an antibody fragment, or any organism, homologue, analog, fusion Physical or functional equivalent. Another aspect of the present invention is to provide an in vitro immunoadsorption kit for specifically adsorbing a specific substance, including: (a) a blood mass separator that separates blood cells and plasma from blood; (b) specific adsorption An in vitro immunoadsorption device for a specific substance, including: (bl) a matrix; and (b2) a specific substance conjugated to the specific substance, which is connected to the matrix or filled in the substrate, and the conjugate The body includes peptides, fragments of peptides or proteins, epitopes, antibodies or antibody fragments, or derivatives, homologues, analogs, fusions or functional equivalents thereof; and (c) reflux devices' It mixes the separated blood cells and the plasma adsorbed by the immunoadsorption device and returns them to the body. ’A In order to make the above and other objects of the present invention 'features and advantages more obvious and easy to understand, the following exemplifies the preferred embodiments and the accompanying drawings to make details

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The explanation is as follows: A brief description of the diagram. Figure 1 is a schematic diagram of the principle and application of the body adsorption device # β ^ 一 立 立 immunosorbent kit of the present invention. ^ The figure shows the flow chart of the construction of AChRa scholar expression vector. Chundi 3 shows the MA of AChR α_ECD and the deduced amino acid sequence. "" When gGIFc forms a fusion protein, an additional linking sequence (GSREF) will be added at 68 8-7202. Section 4 The figure shows the western ink rupture diagram. The three proteins AChR a -ECD-Fe, IgG, and CTLA4-Fc, of which (A) is a non-reduced state treatment, and (B) is a reduced state treatment, with anti-AChR α antibody as Probes to test specific recognition. ^ Figure 5 shows the structure of AChR α-Fc determined by enzyme-linked immunosorbent assay. (A) AChR α — Fc and CTCA4-Fc (1 stomach) Micrograms / ml), Absorbance diagrams obtained by serial dilution of commercially available mouse anti-human ... ⑽ antibodies; (B) Absorbance obtained by combining normal human and patient serum with various dilutions by adding AChR Fc on a micro-quantitative disk. Figure. Detailed description of the invention The structure of the acetylcholine receptor (AChR) is a complex membrane protein formed by α20,000 r (5 subuni t), two α subunits can bind ACh or umbrella nerve Toxin (α-bungarotoxin; a snake venom protein) ° at the extracellular functional site of the α subunit (extracellular do main; ECD) A total of 121 amino acids, of which 67 ~ 76 amino acid sites are most likely to induce the production of antibodies and diseases. This region is called the main immune response.

1238251 _Case No. 89113958_ Year Month and Day _ Zheng V. Description of the invention (5)

Main immuno-genic region (MIR) (Tzartos, SJ et a 1., 1 988, Proc. Natl. Acad. Sci. USA 85: 2899-2903; Barkas, T. et al., 1988 J. Biol. Chem. 363: 5916-5920). In patients with MG, 60% of the anti-A C h R antibodies obtained were identified in or near this region (Lindstrom, J. et a 1., 1 988, Adv. Immunol.

42: 233-284; Schonbeck, S. et al., 1990, Int. Rev. Neurobiol. 32: 1 75-200). Injecting anti-MIR monoclonal antibodies into mice also induces muscle weakness symptoms. Moreover, most anti-MIR antibodies can only recognize the MIR three-dimensional structure (conformation), very few antibodies can recognize MIR on denatured proteins, but their affinity is much lower than the AChR of the natural structure, showing that the ECD of the alpha subunit is MG The key to treatment.

In the present invention, the ECD of the AChR α subunit is genetically fused to the Fc portion of human IgG1. In addition to increasing the solubility, the formed fusion protein can stabilize the structure of AChR α-ECD. In addition, due to the Fc portion, The position of the disulfide bond is retained, and a dimer (AChR a -ECD-Fc) 2 can be formed, similar to the naturally occurring α2 three-dimensional structure, which is easily recognized by the autoantibody and increases the binding force to the autoantibody. . In addition, another advantage of (AChR α -ECD-Fc) 2 is that this fc-containing fusion protein can be purified using an affinity chromatography column of protein A to obtain a high-purity fusion protein. In the present invention, the protein expression system of bacillovirus is selected to express a fusion protein of AChR a -ECD-Fc. This system is a eukaryotic cell virus expression system that infects insects with a recombinant inant baculovirus containing the AChR a -ECD-Fc gene.

0643-5413TWF3; s us anwu.ptc Page 8 1238251 __Case No. 8jj exhibition §8 ___ Month day __- V. Description of the invention (6) Cells can produce a large number of AChR a -ECD-Fc fusion protein. This performance system has the following advantages: (1) can produce a large number of foreign genes; (2) protein modification is similar to mammalian cells; (3) the virus is safe to operate; and (4) the foreign gene protein produced can be Released in culture medium for easy purification. Extracorporeal circulation immunoadsorption device uses different adsorbents to achieve the purpose of immunoadsorption. Early protein A was fixed on agarose (Terman DS et al., J. Immunol., 124: 795, (1 980); New England J. Med., 3 05 ·· 1 1 95 (1 981)), It can specifically adsorb immunoglobulins or immune complexes, but this protein a comes from Staphylococcus aureus (yeU〇w · staphylococcus), which is toxic to the human body. During operation, such as peeling and Into the human body, it is easy to cause injury. Later, Kuroda in Japan used tryptophan or phenylalanine to bond to polyethylene (U.S. Patent No. 4,627,915). This negatively charged surface can adsorb immune complexes, but There is no specificity for harmful antibodies, and some useful proteins will be removed in conjunction. In addition, due to the low concentration of anti-acetylcholine antibodies in the blood, which is in the range of about 100 nM, the protein A currently used in the market is non-specific to all immunoglobulins (about 丨〇mg / ml) into the adsorption, so the column is easy to reach adsorption saturation. For clinical use, it is necessary to install both ||| the pipe column. When one pipe column is adsorbed, the other adsorbed saturated pipe column can be cleaned to facilitate the next adsorption. This repeated cleaning and adsorption is not necessary. economic.

Nakaji, S. et al. (2000) proposed a body for treating myasthenia gravis

1238251 _Case No. 89113958__Year Month Day Amendment ^ __ V. Description of the Invention (7) External immunoadsorption column using the acetylcholine receptor alpha subunit (α183-200) of the synthetic Pacific Electron (Torpedo californica) Covalently immobilized on porous cellulose microbeads to form an adsorbent filled in an adsorption column to 'specifically remove the blocking antibody from the patient's anti-beta biliary receptor antibody (Nakaji et al., 2000, therapeutic Apheresis, 4 (2) ·· 1 2 4-1 26 ·). However, non-naturally derived porous cellulose microbeads have less affinity for autoantibodies than those derived from human peptides. Sano et al. Revealed that the extracellular functional site (ECD) of the recombinant human muscle acetylcholine receptor alpha subunit is a specific junction site for MG autoantibodies (Sano et al.

al., 1991, Int Immunol. 1991 Oct, 3 (10): 983-9). Therefore, in the present invention, a fusion protein derived from the human acetylcholine receptor alpha subunit-extracellular functional site-immunoglobulin fixed region fragment (AChR α-ECD-Fc) is used as a common body, using natural structure. Alpha subunit ECD, and fused with the Fc portion of human IgG1, so that the resulting fusion protein increases solubility, and can more stabilize the AChR a -ECD structure, and because the Fc portion retains disulfide bonds, it can form (AChR α-ECD-Fc) 2 dimer, similar to the naturally occurring α 2 three-dimensional structure, increases the binding force to autoantibodies. In addition, the above combination can be easily purified by protein A affinity chromatography column, which can obtain high Purity fusion protein, and has industrial use value.

In order to solve the disadvantages of the conventional device, the present invention provides an in vitro immunoadsorption device for specifically adsorbing a specific substance, including: (a) a substrate; and (b) a conjugate specifically adsorbed on the specific substance, which is connected to The matrix is or is filled in the matrix. The in vitro immunoadsorption device according to the present invention, wherein the conjugated system means

1238251 Correction number 89m. V. Description of the invention (8) One of two substances that can be combined with each other, including peptides, peptides or protein fragments, epitopes, antibodies or antibody fragments , Or its organism, homologue, analogue, fusion, or functional equivalent. It was fused to the fixed region (Fc) fragment of the immunoglobulin for subsequent purification work. fn, in a preferred embodiment, suitable peptides include any peptide, peptide or protein fragment, antigenic determinant, site, derivative, homologue, analogue, etc. that can be specifically recognized by an autoantibody. Materials, fusions or functional equivalents, such antigen-antibody binding, selection of antigenic sites, etc. are well known to those skilled in the art. In a preferred embodiment of the invention, the peptide comprises an acetylcholine receptor, more preferably an extracellular functional site (ECD) of the acetylcholine receptor. Similarly, the above-mentioned polypeptide can be optionally fused with the fixed region (Fc) fragment of the immunoglobulin, which can increase the solubility of the polypeptide on the one hand, and has the effect of stabilizing its three-dimensional structure on the other hand. According to the present invention, specific substances that can be adsorbed and removed using the device include antibodies (preferably autoantibodies), antigens or harmful substances. Among them, harmful substances generally refer to the health of the human body at the time Examples include, but are not limited to, toxic molecules, low-density lipoprotein cells, very low-density lipoproteins (VLDL), free radicals, viruses, cancer lipopolysaccharide (LPS), bacteria, and the like. The in vitro immunoadsorption device of the present invention can, according to the fact, carry any peptide that can be specifically recognized by the autoantibody to remove the autoantibody in the body. The patient referred to here is, generally speaking, suffering from $ 11 pages 0643-5413TWF3; sus anwu. P t c 1238251

For patients with autoimmune diseases, the sentence »^ u Ren is not limited to 'such as myasthenia f 1, red lupus or rheumatoid arthritis; other diseases' symptoms), as long as it has similarities to autoimmune diseases Due to antibodies

Symptoms that cause disease, I also use the in vitro immunoadsorption device of the present invention to make it carry as much B + B —r-. Xisheng peptide, which can effectively treat or slow down the disease. purpose. ^ Take myasthenia gravis as an example, autoantibodies are produced in the patient, for example, anti-AChR-Ab, and the in vitro immunoadsorption device of the present invention is connected to the acetylcholine receptor Acetylcholine receptor

Extracellular functional sites' and then pass the patient's blood through this device can effectively adsorb pathogenic autoantibodies.

According to the in vitro immunoadsorption device of the present invention, the conjugate can be attached to the matrix or filled in the matrix by an intervening layer or a chemical reaction. Suitable interposers include, but are not limited to, cellulose, agarose, sepharose, dextran, chitin, and chitosan ) And derivatives of the above substances, organic or inorganic porous materials, magnetic beads or microbeads, etc., 'any other interposer conventionally used to bind peptides, antibodies or proteins' includes commercialization Products can be used for the connection purpose of the present invention. On the other hand, a chemical reaction method can also be used to combine the common vehicle and substrate of the present invention, including the conventional cyanogen bromide (CNBr) reaction. In addition, in addition to a general column, the device of the present invention may include, for example, a hollow fiber tube (hoi low fiber). The in vitro immunoadsorption device of the present invention may be combined with other components to form a body.

0643-5413TWF3; s u s anwu.p t c page 12 1238251 case number 89113QSR V. Description of the invention (10) External immunoadsorption kit. Fig. 1 Schematic diagram of Gu Qianshi's principle and application ^ V; hair; ^ in vitro immunoadsorption kit separator and divided into blood 襞 ▲ sphere fluid = outflow: ... pulp-Fc-agarose column, and disease Electro blood: then flow through AChR α ™ ^ Jianshi / β Adsorb anti-AChR antibodies in the patient's plasma into the package: other antibodies and proteins in the plasma will flow out of the d line, and the Λ will be pooled into 6 tubes It can return to the patient's body, which can avoid the abnormal loss of plasma components due to non-specific adsorption. The empty fiber tube bundle 'is not easy to block in the tube bundle due to the large gap of the tube bundle. The plasma separator can also be used without directing blood into the tube bundle to reduce the use of one component. Hereinafter, the present invention will be further described by using specific examples. However, these examples are merely examples and are not intended to limit the purpose of the present invention. Example 1: Construction of AChRa-ECD-Fc fusion protein gene See FIG. 2. A human rhabdomyosarcoma (rhabmyodosarcoma; RD) cell line (CCRC 60113), which can express a large number of acetylcholine receptors, was cultured at a concentration of 10%. Heat deactivated fetal bovine serum (FBS), 2 mmol / L glutamic acid amine, 100 units / ml penicillin, and 100 μg / ml streptomycin in DMEM culture medium at 37 ° C, containing Incubate in a humidified incubator with 5% carbon dioxide. S f 2 1 insect cells were cultured in TNM-FH culture medium (Gibco) containing 10% heat-deactivated fetal bovine blood, and cultured in an incubator at 27 ° C. 1 ml of UltraspecTM RNA (Biotec Lab) lyse RD cells that have formed a single layer in a 35 mm Petri dish, and finely

1238251 ___ 索 号 89113958_ 年月 月 Correction_ V. Description of the invention (11) The lysate is thoroughly mixed. After homogenization, ‘0.2 ml of chloroform’ was added and shaken vigorously for 15 seconds before placing on ice. After centrifugation, carefully transfer the aqueous layer to a clean tube. Add isopropyl alcohol and RNATackTM beads to sink RNA. Finally, purified R N A was precipitated with diethyl pyrocarbonate-treated water (D E P C — w a t e r).

Take 10 micrograms of total RNA and dissolve in 38 microliters of DEPC water. Add 3 μl of DNA primers (100 μg / μl) and mix. The mixture was incubated at 65 ° C for 5 minutes, and then slowly cooled to room temperature to allow the primers to easily attach to the RNA. Add 1 μl of Strata Script RNase H-Reverse Transcriptase and other drugs as mentioned in the operating manual to a total volume of 50 μl. The mixture was incubated at 37 ° C for 1 hour and then 90 ° C for 5 minutes to form the first product. Using 5 microliters of the first product as a template, polymerase chain reaction (PCR) was used to amplify the DNA of the AChR α extracellular functional site (ECD). The primers for this amplification reaction include a forward primer, 5'-CTCGAGACCACCATGGAGCCCTGGCCTCTC-3 'and a reverse primer, 5'-GGATCCCAGGCGCTGCATGACGAAG-3 ,. The PCR conditions were as follows: the first round, 94 ° C, 5 minutes; 54 ° C, 30 seconds; 72 ° C, 2 minutes, and then 2-30 cycles, the conditions were 94 ° C, 30 seconds, 54 ° C, 30 Seconds and 72 ° C, 2 minutes. The final reaction was performed at 72 ° C for 10 minutes.

The PCR fragment was inserted into the PCRII vector, and the correct clone was screened by BamHI enzyme cleavage, and named PCRII / AChR α-ECD. The AChR α fragment was purified by PCRII / AChR α-ECD and digested with BamHI restriction enzyme. The AChR α -ECD fragment was purified and inserted into the improved insect virus expression vector pBachIgGiFc with the human immunoglobulin IgGIFc region.

0643-5413TWF3; susanwu.ptc p.141238251 Case No. 89113958 Amendment V. In the description of the invention (12), the 3'-end of AChR α-ECD and the 5, -end of IgGIFc are combined to generate the pBac- of the present invention. AChR α -ECD-hIgGIFc (Ana-P3) vector. This carrier was deposited at the Strain Center of the Food Industry Development Research Institute of the Republic of China on June 14, 1989, with the deposit number: CCRC 940305. Example 2 · Recombinant protein expression using baculovirus system (1) Production of recombinant virus One hundred and six moth larva cell line S f 2 1 cells were placed in a 35-mm petri dish and cultured overnight. First, gently mix 0.5 micrograms of the pBac-AChR α-ECD-hIgGIFc vector containing the target gene with 100 ng of BacPAK6 viral DNA, then add infectin (Bacfectin), mix and let stand at room temperature for 15 minute. After the serum-containing tnM-FH medium on the overnight culture cells was aspirated, '2 ml of fetal bovine serum-free medium was added and washed twice, and 1.5 ml of fetal bovine serum-free medium was added, and mixed well. Bacfectin reagent and DNA mixture were slowly dripped in and mixed gently, placed in a 27 incubator and reacted for 5 hours. 'Add 1.5 ml of fetal bovine serum-containing medium and place in a 27 ° C incubator for about 72. hour. (2) Determine the presence of recombinant virus

Take 1 microliter of the recombinant virus stock solution and 〇 × cleanser buffer A (detergent buffer A: 50 mM KC1, (K45% Tween 20, 10 mM Tris-HC1 pH 8.4, 0.1 mg / ml) Glycine and 0.45% NP-40) and 10 micrograms of protease κ (Sigma), and add deionized water to a total volume of 100 microliters. After all the reagents are evenly mixed, place them under C for 1 hour. The virus protein is denatured, cut, and then placed in a 100-minute process for 10 minutes to denature the viral DNA. The viral DNA can be placed-it is stored for future use

0643-5413TWF3; s us anwu.p t c

Page 15 1238251 _ Case No. 89113958 _ year month day _ amendment V. Description of the invention (13) Or take 5 microliters for PCR analysis. (3) Plaque assay

Each of the six-well culture dishes was filled with 106 moth larva cell line S f 21 cells and cultured overnight. After removing the TNM-FH culture solution, carefully instill 200 microliters of the virus solution diluted in the culture solution (1 0_4 ~ 1 0 ~ 8) from the center of the culture plate, and carefully maintain the complete monolayer structure of the cells. Incubate at room temperature for 1 hour. Shake the culture plate every 10 -1 5 minutes so that the cells can be completely covered with the virus solution. During the infection, prepare a sterilized 2% low-melting agar gelatin solution and The culture solution of 10% fetal bovine enterprise clear is mixed at an equal volume at 37 ° C. After the virus solution is finished, carefully suck up the virus solution and add 2 ml of agar gelatin mixture solution along the wall of the culture plate. After it was allowed to solidify at room temperature, 1 ml of a culture solution containing 10% FCS was added, and the culture plate was cultured in a 27 ° C incubator for 5 days until virus plaque appeared. For clear virus spots, add 1 ml of a 0.25% (w / v) Neutral Red (Sigma) solution in phosphate buffered saline (PBS) to the agar gelatin layer at 27 ° C Avoid culturing under light for 2 ~ 4 hours. 'Aspirate the supernatant and invert the culture plate to dry overnight.' At this time, it can be seen that the single layer of living cells will be stained with red. .

(4) Magnification of virus viability and concentration (A). Preparation of low-grade virus solution. Put χχ106 into 21-cell cell culture flasks in a cell culture flask of 25 cm 2 overnight. After the culture solution is aspirated, slowly 0.5 ml of the virus solution diluted with the culture solution (the ratio of the virus to the cells is less than 丨) was dripped, and the whole was incubated for one hour at twelve, shaking the culture plate every 10-15 minutes. Aspirate virus solution and add

1238251

5 ml of culture solution, place the culture bottle in a 27 ° C incubator for 5 to 7 days, and wait until the cells are completely infected by the virus and cause cytopathic lysis. Collect the culture solution and store it in 4 ° C and -70 ° Reserve in C and use virus plaque analysis for virus quantification. (B) Preparation of medium concentration virus solution

Put 5 X 106 SF21 cell line in a 75-cm square cell culture flask overnight. After sucking off the culture solution, slowly drip it into a 丨 ml low-dilution disease-free solution (the ratio of virus to cells is less than 1) Incubate at room temperature for 1 hour, and shake the plate several times every 10 to 15 minutes. After removing the virus solution, add 10 ml of the culture solution, and place the culture bottle in a 27 ° C incubator for 5 minutes. ~ 7 days, after the cells are completely infected by the virus and cytopathic lysis occurs, the culture solution is collected and stored at 4 ° C and -70 ° C for later use, and virus plaque analysis is used for virus quantification. Example 3 ·· Expression and purification of AChRa-ECD-Fc fusion protein Sf21 cell line was cultured in a spinner flask (Spinner Hask) from a cell density of 1 to 2 × 10 V ml, and the cells were about 3 to 4 days later Density is 2 X 10V ml, add a virus with the highest protein expression (the ratio of disease to cell is 5 to 10) to infect the cells and continue to culture in a 2 8 ° c incubator for 3 to 5 days. Completely infected with virus and cytopathic lysis

The culture medium was collected, and the protein was purified by protein A affinity chromatography. A human soluble AChR a -Fc fusion protein was produced through the baculovirus system. Since it has a human G1-type immunoglobulin Fc portion, it can be purified using a protein A agar (Pharmacia) column. Protein A Leptin

1238251 _Case No. 89113958 V. Description of the invention (15) Correct the column on the day of the month, pass the protein solution to be purified at 4 ° C with a peristaltic pump to make the binding completely, and then use 10 to 20 times the protein A Wash the gel column column with 50 mM Tris-HC1 buffer at pH 7.0, and finally wash out the protein with 5 times the column volume of μ Glycine-HC1 buffer at pH 3.0. Each 1 ml was collected into a tube, and each microcentrifuge tube already contained 0.1 ml of a 1 M Tris-HCl buffer (pH 9.0). The obtained protein purification solution was poured into a dialysis membrane bag, and the salts were precipitated with 1x PBS buffer. The protein purity was determined by a protein gel, and the protein concentration was quantified by a protein analysis kit (Bio-Rad). 2 // m fiber filter membrane can be used aseptically.

Example 4: Electrophoretic separation and immunochromatographic analysis

The purified protein was separated by electrophoresis with 10% polyacrylamide gel containing sodium lauryl sulfate (SDS). The samples were placed in a solution containing (reduced group) and without (non-reduced group) 0.15M / 3 / 3-mercaptoethanol solution, and were primed at boiling temperature for 5 minutes before adding the colloid. After electrophoresis was completed, Coomassie bri 1 1 iant blue was used for staining. Immunocytosis analysis method is to separate the purified protein by the above SDS-PAGE method, and then transfect the protein onto a nitrocellulose membrane. The membrane was treated with 50 mM Tris-HC1, 0. 15 M NaCl and 5% skimmed milk powder. 1 μg / mL mouse anti-human AChR α antibody (Serotec) was used as a probe. (: Reactions all night 'After washing four times with a buffer containing 1% skimmed milk powder, the membrane was reacted with goat anti-mouse immunoglobulin-bonded horseradish peroxidase. The bound antibodies were made using the ECL system (Amersham) Fu test. The results are shown in Figure 4.

0643-5413TWF3; susanwu.ptc Page 18 1238251 Rev. __Case No. 89113958 V. Description of the Invention (16) Example 5: Identifying AChR a-ECD-Fc (1) Antibody recognizes AChR a-ECD-Fc

The purified AChR a-ECD-Fc or human immunoglobulin (10 µg / ml) was reacted at 4 ° C overnight to be adsorbed on a 96-well microtiter plate. The reaction plate was washed twice with PBS containing 0.05% Tween 20 (PBST), and then PBS containing 10% fetal calf serum was added to each well and reacted in the greenhouse for 2 hours. A 100-fold dilution of mouse anti-human AChR α antibody (Serotec) was added to the first column of micro-quantitative dishes, and diluted with PBS containing 10% fetal bovine serum at a dilution ratio of 1: 3. The total volume in each well was 100 microliters, and incubated at room temperature for 2 hours. The bound mouse anti-human AChR α antibody was measured with amaranth peroxidase-bound sheep anti-ammune globulin (S i gma), and it was treated at 37 ° C for 1 hour, washed six times with PBST, and added 100 micrograms. One liter of ABTS solution was used as a color-developing matrix. After 20 minutes of color development, the absorbance at 405 nm was read by an ELISA reader. ABTS solution: 0.5548 micrograms / ml ABTS was dissolved in ci trie buffer, and then added with 002% H2 02; among them, the citric acid buffer system contained 1.05% citric acid, 1. 42% disodium hydrogen phosphate (Na2HP04), pH 4.0. The results are shown in Figure 5 (A). (2) AChRa-ECD-Fc identified by serum

10 micrograms / ml of AChR α-ECD-Fc or control group fusion protein was adsorbed on a micro-quantitative disk and treated with 10% FBS in PBS for 2 hours before use. Wash twice with PBST. Add blood from MG patients and normal humans to the microplate

It was cleared and diluted with 10% FBS / PBS, and reacted at room temperature for 2 hours. After washing four times with pbsT, 50 microliters of 1: 1 1000 was added in PBS with 10% FBS, and the sheep anti-human / c light key was taken by emulsification. The microtiter plate continues to react at room temperature.

0643-5413TWF3; sus anwu. Ptc P.19 1238251 Case No. 89113958 Amendment 5. Description of the invention (17) should be 1 hour, and then washed with PBST, 100 μL of ABTS color development matrix is added to each well, , Placed in an ELISA reader to read the absorbance at 405 nm. The results are shown in Figure 5 (B). Example 6: Immobilization of AChRa-ECD-Fc 10 mg of AChR α-ECD-Fc prepared in Example 3 was placed in a dialysis bag, and a coupling buffer (coupling buffer; 0.5 M NaCl, 0.1 M NaHC03) was added. (PH 9.0). After dialysis overnight, after replacing the dialysate three times, it was reacted with 4 ml of CNBr-activated colloid (sepharose 4B Fast flow, Pharmacia) on a rotating disk for 2 hours, and the reacted colloid was allowed to stand for several minutes to precipitate. Take a part of the supernatant and measure OD28 (). When OD28Q approaches 〇, the reaction is complete. The colloid was washed with 20 ml of coupling buffer, then 15 ml of 1 M ethanolamine was added, and reacted at room temperature for 2 hours. Unreacted activating groups were blocked, ethanolamine was slowly removed with a filter, and then 0.5 M NaCl, 0.1 Μ sodium acetate buffer, pH 4.0 and coupling buffer 'each 10 times the volume of colloid, wash the colloid three times alternately, each time contact the colloid with the solution for about 5 to 10 minutes, so that the non-covalently bound AChR a -ECD-Fc is released to prepare complete AChR a _ECD-Fc-Sepharose 'stored in 20% alcohol and stored at 4 ° C until use. _ Example 7: Binding capacity of AChR a -ECD-Fc-sepharose to antibody (1) Test for binding ability of anti-AChR α antibody Anti-AChR α monoclonal antibody, after mass production through cell culture, purification with protein A, Then quantify with protein quantification kit (Bio-Rad). Take 100 μl of AChRa-ECD-Fc-agar bran in a 1.5 ml centrifuge tube, wash it 10 times with PBS, and add 200 μg of anti-AChR α antibody at room temperature.

0643-5413TWF3; susanwu.ptc Page 20 1238251 Case No. 89113958 Amendment V. Description of the invention (18) Reaction on a rotating disk for 30 minutes. After the agarose settles, take the supernatant and wash it 10 times with PBS. Finally, The bound anti-AChR α antibody was washed out with 0.1 M citric acid (pH 2.3), and the anti-AChR α antibody content of each part was quantified to evaluate the binding capacity of AChR α -ECD-Fc -sepharose. (2) Binding capacity of anti-AChR α antibody in serum This example simulates the environment of autoantibodies in vivo, and evaluates the ability of the adsorption device of the present invention to remove antibodies. After mixing 24 0/3 0 0 micrograms of anti-AChR α antibody with 20 ml of fetal bovine serum (experimental group) and Tris buffer (control group), the mixture was filtered through a 0.4 5 / zm filter membrane, and the The pump was driven into an AChRa-ECD-Fc-Sepharose column (flow rate 1 ml / min), rinsed with 10 times the volume of PBS, and then the bound anti-AChR α antibody was washed with 0.1 M citric acid (pH 2.3). The concentrations of protein and anti-AChR α antibody were measured separately to determine the true binding ability of AChR a -ECD_Fc-sepharose when the serum passed. The results are shown in Table 1. Table 1: Comparison of antibody adsorption in serum and buffer. Note · FBS: fetal calf serum; Tris: hydroxymethylaminomethane buffer. mm Machine Lay Knot Every Attendance AtfrAChR ^ S tmi m 74 lffi © 1 skim 2 m 91 W 62 15 & 5 ΰ5.5 FREE ArtAChRij / Tris tmi 300 23 277 923 tmi 300 54 S20 261t22 87.tt7.3 Results:

0643-5413TWF3; susanwu.ptc Page 21 1238251 tm 89113958 Date of the invention (19) RD is a human rhabdomyosarcoma cell that can express acetylcholine receptors. The information obtained from Gene Bank was used to design primers for PCR. The extracellular functional sites of AChR α were selected by TA colonization method (Invi trogen), and the jaundice was selected into the corpse 〇1? 11 vector, human (:} 11? The gene of 〇:-£ 〇〇 is 735. From the gene sequence analysis of PCRI I / AChR α-ECD, two different sequences of AChRa-ECD are obtained, one is missing the 43th amino acid (valine acid ) (Figure 3) 'The other is to insert a 25 amino acid sequence at the 59th amino acid position. Because of the consideration of maintaining the stereo structure of AChR a, the first sequence is selected as the later The basis of the experiment. After that, AChR a -ECD was cloned into pBachIgGIFc to produce pBac-AChRa-ECD-hIgGIFc plastids, which can be homologously recombined with Bac 6 linear virus DNA to produce recombinant viruses. Infection of linear viral DNA with pBac-AChR (transfectin) at the same time, 3 days later, the cells were infected with the virus and caused lesions. The supernatant was collected and immunoprecipitated with protein A-agar. Analyzed by SDS-PAGE, one can be seen at the position of molecular weight 53 kDa. The protein band is estimated to be AChR α-ECD-Fc. Repeated infection of the virus supernatant can amplify the virus concentration, analyze the virus titer with a plaque, and pick out a single plaque to confirm the recombinant virus with PCR. A large amount of AChR α-ECD-Fc was obtained. The present invention produced AChRa-ECD-Fc in a 1-liter rotating culture flask with a viral infection amount of about 5 to 10 (M · 〇 · 2: 5 to 10), and the yield was three days after infection. Approximately 2 μg / ml, the supernatant was collected, filtered, and then passed through a protein A-agar column at a flow rate of 1 ml / min. After washing the column with PBS, it was washed with 0.1 M glycine ( (pH 2 · 3)

0643-5413TWF3; susanwu.ptc Page 22 1238251 Case No. 89113958 Amendment V. Description of the invention (20) AChR a -ECD-Fc precipitated, after dialysis with PBS, concentrated filtration, and stored at-20 C. The purified AChRa-ECD-Fc was confirmed by Western blotting method. In the reduced and non-reduced state, it was detected with a commercially available anti-AChR antibody. The result was 53 kDa in the reduced state, but not in the reduced state. (non-reduced) is 100 kDa (see Figure 4), showing that the AChR α-ECD-Fc expressed by the present invention will form a dimer, which is similar to the natural structure of the acetylcholine receptor (/ 3 r 5), which is beneficial for binding anti-AChR antibodies. AChR a-ECD-Fc and CTLA4-FC (a cell surface antigen) were combined in 96-well culture plates and detected with anti-human AChR antibodies. Figure 5 (A) shows that the anti-human AChR antibody can only specifically recognize AChR α-ECD-Fc 'and cannot recognize unrelated CTLA4-Fc protein, showing that the AChR a-ECD-Fc expressed by the present invention still retains Its natural structure. Similarly, the serum of patients with myasthenia gravis and normal subjects can still be found, but the serum of patients has obvious binding ability, while the serum of normal subjects has lower background values (see Figure 5 (B)). In combination with the above-mentioned AChR a -ECD-Fc expressed in the present invention, it is effective to bind an anti-AChR antibody in a patient with an autoimmune disease. The coupling reaction of 10 mg of AChR a -ECD-Fc and 4 ml of CNBr-activated adipose sugar was used to produce AChR a -ECD-Fc-sepharose, and the amount of uncoupled AChR α-ECD-Fc was estimated. The coupling rate is about 85%. In order to evaluate the adsorption efficiency of AChR a-ECD-Fc on the antibody after coupling, 1 ml of AChRα-ECD-Fc-sepharose was taken out and mixed with 300 μg of anti-AChRα antibody to test its anti-AChR a antibody binding capacity. Unbound 11 11 BH 1 ilii 111 1! 1 after analysis 1 _ __ 0643-5413TWF3; susanwu.ptc p. 231238251 _ case number 89113958 _ year and month correction _ 5. Description of the invention (21) The antibody is 50 micrograms, showing The binding force of 1 ml of AChRa-ECD_Fc-sepharose was 250 micrograms. In order to test whether AChR a -ECD-Fc-agar bran can adsorb anti-AChR α antibodies in blood, a fixed amount of anti-AChR α antibodies was added to fetal bovine serum to simulate the feasibility of this device in practical application. . 20 ml of fetal bovine serum mixed with anti-AChR α antibody was pumped into a 1 ml AChR a -ECD-Fc -sepharose column. Then, the amount of anti-AChR α antibody before and after the device was introduced was measured by ELISA method. Table 1 shows that about 65% of the antibodies have been adsorbed on the device before and after the comparison, showing that this device can indeed be used to remove anti-AChR α antibodies in the blood. At the same time, Bion compared the binding capacity of anti-AChR α antibodies in general buffers, of which about 87% of the antibodies were adsorbed, indicating that even in the presence of serum, the adsorption of AChR α -ECD-Fc-sepharose anti-AChR α antibodies It has not changed much and still retains about 75% adsorption capacity. Therefore, it can be applied to myocardial helmets and people to achieve therapeutic effects. ~ Although the present invention has been disclosed as above with a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with the art can make various changes and decorations without departing from the spirit of the present invention. The scope of protection of an invention shall be determined by the scope of the attached patent application. W

Claims (1)

1238251.. ', Case No. 89113958 ..... Amendment_ VI. Patent application scope 1. An in vitro immunoadsorption device for specific adsorption of antibodies, comprising: (a) a matrix; and (b) specific The conjugate that is adsorbed to the antibody is linked to the matrix or filled in the matrix; wherein, the conjugated system acetylcholine receptor alpha subunit-extracellular functional site-immunoglobulin fixed region fragment (AChRa-ECD-Fc) fusion protein. 2. The in vitro immunoadsorption device according to item 1 of the scope of the patent application, wherein the co-existence system is generated by the pBac-AChR a-ECD-hlgGlFc vector through the baculovirus expression system, and the above vector has been in the Republic of China in 1989. On June 14th, it was deposited at the Strain Center of the Food Industry Development Research Institute of the Republic of China under CCRC 940305. 3. The in vitro immunoadsorption device according to item 1 of the patent application scope, wherein the antibody is an autoantibody. 4. The in vitro immunoadsorption device according to item 3 of the scope of patent application, wherein the autoantibody system produces antibodies produced by patients with autoimmune diseases. 5. The in vitro immunoadsorption device according to item 4 of the scope of the patent application, wherein the autoimmune disease is myasthenia gravis. 6. The in vitro immunoadsorption device according to item 1 of the scope of the patent application, wherein the conjugated system is connected to the substrate or filled in the substrate through an intermediate interlayer or a chemical reaction. 7. The in vitro immunoadsorption device according to item 6 of the scope of the patent application, wherein the intermediate interlayer comprises agar, agarose, dextran, cellulose, chitin, chitosan and derivatives of the above substances, Organic or inorganic porous 0643-5413TWF3; susanwu.ptc Page 25 1238251 _Case No 89Π3958_Year Month Day__ VI. Scope of patent application Material, magnetic beads or micro beads. 8. The in vitro immunoadsorption device according to item 6 of the scope of the patent application, wherein the chemical reaction includes a linking reaction of cyanogen bromide. 9. The in vitro immunoadsorption device according to item 1 of the patent application scope, wherein the device is a column or a hollow fiber tube bundle. 10. An in vitro immunoadsorption kit for specific adsorption of antibodies, including: (a) a plasma separator that separates blood cells and plasma from blood; (b) an in vitro immunoadsorption device that specifically adsorbs antibodies, Including: (b 1) a matrix; and (b 2) a conjugate specifically adsorbed to the antibody, which is linked to the matrix or filled in the matrix; wherein the co-system bile receptor α Subunit-extracellular functional site-fusion protein of immunoglobulin fixed region fragment (AChR α-ECD-Fc); and (c) a reflux device that mixes the separated blood cells and the plasma adsorbed by the immunoadsorption device and sends them Back inside. 1 1. The in vitro immunoadsorption kit according to item 10 of the patent application scope, wherein the co-car system is generated by the pBac-AChRa-ECD-hIgGIFc vector via the baculovirus expression system, and the above vector has been obtained in the Republic of China 8 On June 14th, 9th, it was deposited in the Strain Center of the Food Industry Development Research Institute of the Republic of China with the deposit number CCRC 940305. 1 2. The in vitro immunosorbent kit according to item 10 of the patent application scope, wherein the antibody is an autoantibody. 1 3. In vitro immunosorbent sleeve as described in item 12 of the scope of patent application 0643-5413TWF3; susanwu.ptc Page 26 1238251 Case No. 89113958 Amendment VI. Patent application group, in which the antibodies produced by patients with autoimmune diseases of the autoantibody system1. The in vitro immunoadsorption kit is described, wherein the autoimmune disease is myasthenia gravis. 0643 -5413TWF3; s us anwu.p t c page 27