WO2003084998A1 - Anticorps monoclonal neutralisant la megsine - Google Patents

Anticorps monoclonal neutralisant la megsine Download PDF

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Publication number
WO2003084998A1
WO2003084998A1 PCT/JP2003/004459 JP0304459W WO03084998A1 WO 2003084998 A1 WO2003084998 A1 WO 2003084998A1 JP 0304459 W JP0304459 W JP 0304459W WO 03084998 A1 WO03084998 A1 WO 03084998A1
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megsin
monoclonal antibody
antibody
variable region
ms18a
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PCT/JP2003/004459
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English (en)
Japanese (ja)
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Kiyoshi Kurokawa
Toshio Miyata
Takeshi Chisaka
Kazuya Kunitou
Masahiro Asakura
Yumi Hirooka
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Fuso Pharmaceutical Industries, Ltd.
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Priority to JP2003582192A priority Critical patent/JP4225487B2/ja
Priority to AU2003236346A priority patent/AU2003236346A1/en
Publication of WO2003084998A1 publication Critical patent/WO2003084998A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to a monoclonal antibody that selectively binds to a specific serpin (serine protease), namely, a reactive loop region of megsin, a method for producing the same, and mesangial proliferation using the monoclonal antibody.
  • the present invention relates to a composition for treating and / or preventing a composition for the treatment and / or prevention of glomerulonephritis.
  • Renal failure is a condition that ultimately leads to kidney disease patients.
  • causes and backgrounds are not uniform, and renal disorders occur due to non-renal lesions such as drug addiction, infectious diseases, malignancies, diabetes mellitus, and systemic lupus erythematosus (SLE). Be looked at.
  • Kidney transplantation is the only treatment for end-stage renal failure in which no renal hemofiltration or detoxification functions.However, in Japan, it is difficult to say that the supply system for transplanted kidneys is sufficiently developed. . In addition, social recognition of transplantation therapy itself has not advanced. The number of renal transplant cases in Japan is only about 700 cases a year, and this figure has not increased in recent years. Therefore, at present, dialysis is the only renal replacement therapy. Currently, Japan has an estimated 210,000 end-stage renal failure dialysis patients, and is the world's number-one patient per population. The average cost of treatment per person is about 6 million yen per year, which is one of the major causes of pressure on the medical insurance system.
  • mesangial cells are organ-specific cells found only in the kidney and play an important role in maintaining the structure and function of renal glomeruli.
  • mesangial cells are organ-specific cells found only in the kidney and play an important role in maintaining the structure and function of renal glomeruli.
  • the proliferation of mesangial cells themselves and an increase in extracellular matrix secreted by mesangial cells are observed, and it is speculated that these cells are deeply involved in the onset and progression of disease. From these facts, it is considered indispensable to clarify the biological characteristics of mesangial cells in order to elucidate the mechanism of glomerular damage.
  • the specificity at the gene level for mesangial cells has not been determined.
  • the present inventors clarified the gene expression pattern of mesangial cells and attempted to analyze the cell characteristics at the gene level.
  • the present inventors extracted mRNA from cultured human mesangial cells and prepared a 3′-directed cDNA library. Large-scale DNA sequencing and overnight analysis of the cloned gene fragments were performed [Yasuda, Y. et al .: Kidney Int., 53: 154-158, 1998]. As a result, a gene consisting of 2,249 bp in length, named megsin, was isolated as a gene that is particularly strongly expressed in mesangial cells.
  • megsin protein consisting of 380 amino acids, encoded by the full-length cDNA clone of megsin.
  • an amino acid homology search by the FASTA program was performed using the SwissProt amino acid sequence database.
  • the serine protease inhibitor (serpin: SERPIN) superfamily [Carrel 1, RW et al .: Trends Biochem. Sci., 10:20, 1985; Carrel 1, RW et al. : Cold Spring Harbor Symp. Quant. Biol., 52: 527, 1987; Kruithof, EK0. Et al .: Blood, 86, 4007, 1995; Pote immediately a, J.
  • a consensus sequence (EEGTEMAAT) in the reactive loop site which is important as a bioactive central site. It was found that a sequence (EEGTEATAAT / SEQ ID NO: 3) similar to / SEQ ID NO: 2) was present. That is, megsin has structural characteristics of serpin, and has a reactive loop region (P17-P5 ′: EEGTEATAATGS NIVEKQLPQS / SEQ ID NO: 1) which is an active site similarly to other serpins [Miyata, T. et al. et al .: J. Clin. Invest., 102: 828-836, 1998] o
  • Serpins include antithrombin and plasminogen activator inhibitors
  • serpin plasminogen activator inhibitor: PAD, ovalbumin, etc. are known. These serpin molecules have high homology to each other, and their three-dimensional structures are very similar. Serpin is a target serine protease It is known that the enzyme forms a complex with the enzyme and inhibits the enzymatic activity It is the flexible reactive loop site of servin that binds to the active site of the protease.
  • the serpin-type fold consists of three antiparallel parts, partially covered by a helix; a dense structure created by six seas A, B, and C.
  • the uncleaved ovalbumin structure is considered to be the canonical form of serpin, with sheet A containing five strands.
  • the flexible loop starts at the end of strand 3 of sheet 3 A, followed by the a helix on the outside of the molecule, followed by the strand (] 316) at the end of sheet 3/3, and contained in sheet 3 Reach the starting point of the strand (/ 317).
  • the flexible loop region of activated antithrombin is in the same general position as ovalbumin, but some residues close to the beginning of the loop are located between
  • the loop region protrudes from the main part of the serpin molecule so that it can bind to the active site of serine protease (FIG. 1 (a)).
  • the latter half of the loop region occupies almost the same position as in active antithrombin.
  • the two new ends created by the fragmentation are at both ends of the molecule, about 70 A apart.
  • the j3 strand is added to the / 3 sheet A, similar to the truncated form of ⁇ antitrypsin, but the rest of the flexible loop region forms a loop outside the molecule.
  • 3 sheet B is connected to the i3 strand, and ⁇ sheet C has no end strand.
  • the loop region which is the active site of serpin, can take three forms: “active”, “cut”, and “depressed”.
  • the loop changes to a long 0-strand and is inserted in the center of the ⁇ -sheet.
  • Adjacent strands in the j8 sheet must first be separated in order to make a major structural change, such as inserting a jS strand into a stable / 3 sheet already configured. This means that not only do many of the hydrophobic contacts created inside the molecule become tightly packed, but many of the hydrogen bonds break. Newly formed hydrogen bonding and filling contacts must be recreated after the extra; 8 strand is inserted.
  • Such large changes in the / 3 structure were not expected at all before the determination of the structure of serpin, and have not yet been observed in other systems [Branden, C. et al.-. Introduc t ion to Prote in Structure Second Edition, 1999]. Disclosure of the invention
  • An object of the present invention is to provide a megsin-neutralized antibody which is effective for treating diseases associated with mesangial cells. Further, the present invention provides a monoclonal antibody that selectively binds to a reactive loop region of megsin, a method for producing the same, and a method for treating mesangial proliferative glomerulonephritis using the monoclonal antibody and for preventing or preventing Z or prophylaxis. It is an object to provide a composition.
  • Megsin has the structural characteristics of serine protein inhibitor (SERPIN), and has a reactive loop region (P17-P5 ': EEGTEATAATGSNI VEKQLPQSZ SEQ ID NO: 1) which is the active site like other serpins.
  • SERPIN serine protein inhibitor
  • P17-P5 ' EEGTEATAATGSNI VEKQLPQSZ SEQ ID NO: 1
  • mice with megsin show progressive mesangial substrate expansion, mesangial cell proliferation, and an increase in immune complex deposits, suggesting that nephritis is likely to be induced by their servin activity. Suggested [Miyata, T. et al .: J. Clin. Inves t., 109, 585, 2002].
  • megsin is a high glomerular expression gene belonging to the serpin superfamily, this gene is a functional gene in the glomerulus and is considered to be related to the development of glomerulonephritis Z. And, since the megsin protein has a reactive loop site unique to the serpin superfamily, it is considered that it acts as a suppressor for certain serine proteases.
  • Protease activity is kept constant by the balance between the activity of the protease and its inhibitor, and it is known that fluctuations thereof have a significant effect on tissue / cell functions and disease states. In fact, during glomerular sclerosis, the degradation of extracellular matrix proteins by proteases is observed.
  • mice expressing high levels of PAI-1 a kind of serpin (27.7% amino acid homology with megsin protein), were Studies show that bleomycin-reactive fibrosis is induced more frequently than wild mice, but that fibrosis is less likely to occur in PAI-1 deficient mice.
  • serpins not only suppress serine oral protease activity but also participate in blood coagulation, fibrinolysis, inflammatory response, cell differentiation, proliferation, and apoptosis, and their physiological mechanisms and pathophysiological significance are diverse.
  • megsin has a biologically important effect on the function of mesangium. .
  • single genetic manipulation of megsin can generate early mesangial lesions present in experimental and human glomerulonephritis.
  • megsin is also involved in the development of mesangial proliferative glomerulonephritis in animal individuals.
  • the present inventors have scrutinized antibodies having neutralizing activity against megsin using recombinant megsin, and found that a monoclonal antibody recognizing a specific region can neutralize the activity of megsin.
  • the present invention has been completed.
  • Monoclonal antibodies against megsin are known (W00 / 57189), but specifically bind to the epitope in the reactive loop region of megsin (P17-P5 ': EEGTEATAATGSNIVEKQLPQSZ SEQ ID NO: 1). No knowledge of the antibody was confirmed. Also, no antibody that neutralizes the action of megsin was known.
  • the monoclonal antibody according to the present invention can neutralize the reaction between megsin and plasmin, one of the ligands for megsin. That is, the present invention relates to the following monoclonal antibodies and uses thereof.
  • a monoclonal antibody having a megsin neutralizing activity [1] A monoclonal antibody having a megsin neutralizing activity.
  • a method for producing a monoclonal antibody comprising a step of culturing hybridoma MS18a deposited as FERM BP-8349, and recovering immunoglobulin contained in the culture.
  • a composition for the treatment and / or prevention of mesangial growth and glomerulonephritis comprising an effective amount of any of the following components (a) to (d).
  • a method for neutralizing the activity of megsin comprising a step of bringing the component according to any of the following (a) to (d) into contact with megsin:
  • the present invention relates to a method for treating and / or preventing mesangial proliferative glomerulonephritis, which comprises a step of administering an effective amount of the component according to any of the above) to (d). Furthermore, the present invention relates to the use of the component according to any one of the above (a) to (d) in the manufacture of a composition for treating and / or preventing mesangial proliferative glomerulonephritis.
  • the present invention provides a monoclonal antibody that neutralizes the action of megsin.
  • neutralizing the action of megsin means the action of neutralizing the serpin activity of megsin.
  • Serpin activity is an inhibitory effect on serine protease protease activity.
  • neutralization includes not only complete suppression of serpin activity but also partial suppression.
  • plasmin can be used as a serine protease, and its neutralizing activity can be confirmed using the inhibitory effect of the monoclonal antibody on the inhibition of the protease activity by megsin as an index.
  • Protease activity can be measured, for example, by observing the digestion of the fluorescent substrate.
  • the present inventors have found that a monoclonal antibody that recognizes a reactive loop region of megsin has a neutralizing effect on megsin. That is, the present invention relates to the reaction of megsin.
  • the reactive loop region of megsin refers to a region in the amino acid sequence of megsin, particularly the region consisting of the amino acid sequence of SEQ ID NO: 1. Therefore, the monoclonal antibody of the present invention is a monoclonal antibody that recognizes an epitope constituted by a peptide having the amino acid sequence of SEQ ID NO: 1.
  • a monoclonal antibody recognizing the amino acid sequence NIVEKQ (SEQ ID NO: 4) is a desirable monoclonal antibody capable of effectively suppressing the serpin activity of megsin.
  • the monoclonal antibody recognizes the epitope composed of the peptide having the amino acid sequence of SEQ ID NO: 1.
  • peptides having various amino acid sequences are used to identify epitopes recognized by the monoclonal antibody.
  • a monoclonal antibody against megsin can be obtained by a known method using human megsin or its domain peptide as an immunogen. The method for obtaining the monoclonal antibody will be specifically described later.
  • the monoclonal antibody of the present invention can be obtained by selecting the obtained monoclonal antibody based on the above evaluation method.
  • hybridoma Ms18a cell As a hybridoma producing the monoclonal antibody of the present invention, for example, a hybridoma Ms18a cell can be shown.
  • the hybridoma Ms18a is a hybridoma that produces a monoclonal antibody that recognizes NIVEKQ (SEQ ID NO: 4) in the amino acid sequence constituting the reactive loop of megsin.
  • the monoclonal antibody Ms 18a produced by the hybridoma Ms 18a has a megsin neutralizing activity.
  • Hypri-Dorma Msl8a was established on April 1, 2002 (Hara Deposit Date) at the Patented Depositary Center of the National Institute of Advanced Industrial Science and Technology located at 1-1, Higashi 1-1, Tsukuba-shi, Ibaraki, Japan. In contrast, it has been deposited under accession number FERM BP-8349.
  • the isotype of the monoclonal antibody produced by this hybridoma Ms18a cell line was IgGl for the H chain and ⁇ for the L chain.
  • the present invention also provides a class switch mutation of the above antibody. , Such as isotypes IgG3, IgGK IgG2b, IgG2a and other variants belonging to the immunoglobulin subclass, and such variants can be made by the method of Martin et al. in, C. et al .: J. Immunol. Methods., 145: 1118, 1991).
  • the production of monoclonal antibodies to megsin requires megsin, which can be used as an immunogenic antigen.
  • Megsin as an antigen can be obtained using cultured cells, for example, megsin producing cells.
  • megsin-producing cells include, for example, cells derived from human kidney.
  • the megsin-producing cells are cultured using a medium or culture method known in the art or substantially similar thereto, and the megsin produced in the culture supernatant is subjected to, for example, ion exchange chromatography and ion exchange chromatography. It can be purified by affinity chromatography using a polyclonal antibody. Recombinant megsin can also be used.
  • host cells are transformed by a recombinant vector containing a gene fragment containing the nucleotide sequence encoding the amino acid sequence of megsin, and the transformed host is cultured to contain the amino acid sequence of megsin.
  • the recombinant vector containing the megsin cDNA is prepared by a conventional gene recombination technique, for example, by inserting it into a plasmid vector.
  • a virus such as vaccinia virus or paculovirus can be used in addition to plasmid phage.
  • prokaryotes such as Escherichia coli, Bacillus subtilis, and actinomycetes, and various cells, for example, commercially available cell lines such as animal cells and CH0 cells, and eukaryotes such as yeast, plant cells, and insect cells. it can.
  • a promoter that can be used for prokaryotes for example, tributophan synthase operon, lactose operon, and the like can be used.
  • Promoters that can be used in eukaryotes include, for example, viral promoters, promoters for alcohol dehydrogenase, promoters for glycolytic enzymes, and the like.
  • the megsin thus prepared may be further used as an immunogenic conjugate, but can be used as it is for immunizing an animal by mixing it with an appropriate adjuvant.
  • antigens can be obtained from various raw materials, such as cultured cells, cultured tissues, and transformed cells, using known methods for producing antigens, such as salting-out such as ammonium sulfate precipitation, and gel filtration chromatography using Sephadex. Purification by ion exchange chromatography, hydrophobic chromatography, dye gel chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography, high performance liquid chromatography, etc. Can be obtained.
  • megsin is obtained by fragmenting the megsin, or by selecting a characteristic sequence region based on the amino acid sequence deduced from the cloned and sequenced cDNA sequence, designing the polypeptide, and chemically synthesizing it. May be combined with various carrier proteins via an appropriate condensing agent to form a hapten-protein immunoconjugate, and using this, only a specific sequence can be used. Recognizable monoclonal antibodies can also be designed. A cysteine residue or the like can be added to the designed polypeptide in advance so that the immunogenic conjugate can be easily prepared.
  • the present invention provides at least one monoclonal antibody that specifically binds to megsin.
  • the monoclonal antibody according to the present invention is obtained by immunizing an animal using recombinant megsin as an immunogen, cell fusion between myeloma cells and antibody-producing cells, selection and monocloning of a hybridoma, production of a monoclonal antibody, if necessary. It can be made by a process such as ascites.
  • the animal is immunized, for example, as follows.
  • Human megsin protein purified according to a known method [Miyata, T. et al .: J. Clin. Invest., 120: 828, 1998] is immunized to mammals such as rats and mice.
  • Mammalian animals have a permanent proliferative potential during cell fusion It is preferable to use animals of the same strain as the cells.
  • the age of the animal is preferably 8 to 10 weeks for mice, for example. Sex may be either male or female.
  • human immunoglobulin By using a transgenic animal in which the immunoglobulin gene has been modified into a human gene as an immunized animal, human immunoglobulin can also be produced.
  • a method for obtaining an antibody having the desired reactivity using a transgenic animal in which the immunoglobulin gene has been modified into a human gene is known. While the immunoglobulin thus obtained can be obtained from animals, the method of complete immunization can be performed by purifying purified human megsin protein with an appropriate adjuvant (for example, Freund's complete adjuvant or aluminum hydroxide gel-pertussis). Vaccination, etc.) and then subcutaneously, intraperitoneally, or intravenously into animals. Thereafter, this immunization operation is performed 2 to 5 times at an interval of 1 to 2 weeks. Final immunization is performed by intraperitoneally administering 0.5-2 x g human megsin protein to the animal.
  • an adjuvant for example, Freund's complete adjuvant or aluminum hydroxide gel-per
  • a polyclonal antibody is obtained from the body fluid of the immunized animal. Blood is collected from the fundus venous plexus 3 to 7 days after each immunization, and the antibody titer of the serum is measured. When the antibody titer is sufficiently raised, the antibody or antibody-producing cells are collected.
  • the antibody titer to megsin can be measured by a technique such as ELISA.
  • the ELISA for measuring the antibody titer can be carried out by adding serum to a plate coated with megsin, and further adding a labeled antibody against IgG of the immunized animal.
  • the adjuvant used with the antigen examples include Freund's complete adjuvant, Haiti adjuvant, pertussis vaccine, BCG, ribosome, aluminum hydroxide, silica gel and the like.
  • animals such as mice such as Balb / c mice and FI mice can be used.
  • Antibody-producing cells are collected from animals immunized with the human megsin protein as described above.
  • Antibody-producing cells can be obtained from spleen, lymph nodes, peripheral blood, etc., with spleen being particularly preferred.
  • MEM Essential Medium
  • the spleen is aseptically removed and After shredding in Essential Medium (MEM) medium (manufactured by Nissui Pharmaceutical Co., Ltd.), dissociating with forceps, centrifuging at 1,200 rpm x 5 minutes, removing the supernatant, and removing the Tris-HCl buffer (PH 7.65) for 1 to 2 minutes to remove red blood cells, and then wash three times with MEM medium to obtain spleen cells for cell fusion.
  • MEM Essential Medium
  • the tumor cell line used before cell fusion can be selected, for example, from a cell line that does not produce immunoglobulin.
  • the permanently proliferating cell to be fused any cell having permanent proliferation can be used, but myeloma cells (myeloma) are generally used. It is desirable to use permanently proliferating cells derived from the same animal species as the antibody-producing cells.
  • mice the following cell lines are known as bone tumor cell lines derived from 8-azaguanine-resistant mice (Balb / c).
  • P3-X63Ag8-Ul P3-U1 [Current.Topics in Microbiol. Immunol., 81: 1, 1978]
  • P3 / NSl / l-Ag4-l NS-1 [Eur. J. Immunol., 6: 511, 1976]
  • These immortalized cell lines include 8 Azaguanin medium (RPMI-1640 medium Darutami emissions (1. 5 mM), 2_ mercaptoethanol (5 X 10- 5 M), Gen evening mycin (10 xg / mL) and ⁇ Subculture with normal medium supplemented with fetal serum (FCS, CLS) (10%) and 8-azaguanine (15 xg / mL) and 3-4 days before cell fusion The cells are passaged to a normal medium at the end of the fusion, and the number of cells is 2 x 10 7 or more on the day of fusion.
  • Cell fusion between an antibody-producing cell and a myeloma cell is performed, for example, as follows.
  • the antibody-producing cells and the permanently proliferating cells described above are thoroughly washed with a MEM medium or PBS, and mixed so that the cell number becomes 5 to 10: 1.
  • Polyethylene glycol Ichiru as agent -.. 1000 (PEG- 1000) l ⁇ 4g, dimethyl sulfoxide 0.5 to 1 as MEM medium l ⁇ 4mL and cell fusion promoter OML mixture 0.1 to 1 of OmL / ⁇ 8 pieces Cells are added to cause cell fusion.
  • the suspension was dispensed by 100 L / wel 1 the culture plate for 96 Ueru, in 5% C0 2 Inki Yube Isseki primary, cultured for 3-5 days at 37 ° C.
  • 100 L / we 11 HAT medium medium supplemented with hypoxanthine ( ⁇ -' ⁇ ) and thymidine (1.5 ⁇ 10—3 ⁇ 40 and aminobuterin (4 ⁇ 10—3 ⁇ 4) in a normal medium
  • further culturing 3 days. to remove half volume of the culture supernatant for each subsequent 3 days, newly same amount of HAT medium was added, in 5% C0 2 incubator primary, cultured for about 2 weeks at 37 ° C.
  • a hybridoma culture supernatant is added to a solid phase on which a megsin protein antigen is adsorbed directly or together with a carrier, and then an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme is added, and the label is added.
  • the antibody titer can be measured.
  • a microplate or the like is used for the solid phase.
  • an anti-mouse immunoglobulin antibody is used as the anti-immunoglobulin antibody.
  • protein A can be added to detect an anti-megsin protein monoclonal antibody bound to a solid phase.
  • the hybridoma culture supernatant was added to the solid phase to which the anti-immunoglobulin antibody or protein A was adsorbed,
  • the antibody titer can also be measured by adding a megsin protein labeled with a substance or enzyme.
  • Cloning was repeated four times by limiting dilution for the wells in which the production of antibodies reactive with megsin protein was observed, and those showing a stable megsin protein antibody titer were produced as anti-megsin protein monoclonal antibodies. Select as a hybridoma strain.
  • Monoclonal antibodies are produced by culturing the hybridomas obtained as described above in U / F in vitro.
  • the desired monoclonal antibody can be cultured in an appropriate medium such as a MEM medium containing FCS or RPMI-1640 medium and obtained from the culture supernatant.
  • Culture of the hybridomas in] ⁇ // is preferably performed in a serum-free medium, giving an optimal amount of antibody to the supernatant.
  • the hybridoma When culturing in r / ra, transplant the hybridoma into any animal.
  • a host animal for transplantation it is preferable to use an animal of the same species as the animal from which the spleen cells used for cell fusion have been collected.
  • 8 to 10-week-old Balb / c female mice treated with pristane are intraperitoneally administered with 2 to 4 ⁇ 10 6 cells / animal of the anti-megsin protein monoclonal antibody-producing hybridoma cells obtained above.
  • the pristane treatment is performed, for example, by intraperitoneal administration of 2,6,10,14-tetramethylpentyldecane-pristane-0.5 mL and rearing for 2 weeks.
  • ascites containing a high concentration of monoclonal antibody accumulates in the abdominal cavity of the mouse and the abdomen grows.
  • the ascites is collected from the mouse, centrifuged (3, O0OrpmX for 5 minutes) to remove solids, and IgG is purified.
  • the ascites and culture supernatant are salted out using 50% ammonium sulfate and dialyzed against PBS for 1-2 weeks.
  • the dialyzed fraction is passed through a protein A sepharose column, and the IgG fraction is collected to obtain a purified monoclonal antibody.
  • This monoclonal antibody can neutralize the reaction between megsin and plasmin, one of the ligands for megsin
  • the antibody isotype can be obtained from a commercially available kit (Gibco BRL, Mouse Antibody Isotyping Kit et al.) Or by the Okuiguchi Nii (double immunodiffusion) method (Introduction to Immunology Experiments, Biological Chemistry Experiments 15, Kagaku Shuppan Sen-yu, p. 74, 1981) .
  • the protein mass is calculated by the Folin method and the absorbance at 280 nm (1.4 (0D 280 ) immunoglobulin lmg / mL).
  • ascites of hybridomas can be used.
  • each hybridoma is transplanted into the abdominal cavity of a histocompatibility animal that is syngeneic to the myeloma cell-derived animal and allowed to proliferate, or each hybridoma is transplanted into a nude mouse or the like and is produced in ascites.
  • a monoclonal antibody can be obtained.
  • Animals can be given a mineral oil such as pristane intraperitoneally prior to transplanting the hybridoma.
  • the ascites fluid can be purified as it is or by a conventional method. For example, purification by salting out such as ammonium sulfate precipitation method, gel filtration method using Sephadex, etc., ion exchange chromatography, electrophoresis, dialysis, ultrafiltration method, affinity chromatography, high-performance liquid chromatography, etc. can do.
  • the characteristics of the monoclonal antibody obtained as described above can be determined by, for example, enzyme immunoassay (ELISA).
  • the monoclonal antibody of the present invention can specifically bind to the reactive loop region of human megsin and neutralize the reaction between megsin and plasmin, one of the ligands for megsin. Therefore, the monoclonal antibody of the present invention is useful as a method for neutralizing the activity of megsin or a neutralizing agent for the activity. That is, the present invention provides a method for neutralizing the activity of megsin, which comprises a step of bringing the component described in any of the following (a) to (d) into contact with megsin: Alternatively, the present invention relates to a megsin activity neutralizer containing, as an active ingredient, any one of the following (a) to (d):
  • megsin has an effect of inducing the proliferation of mesangial mums cells. Since the monoclonal antibody of the present invention neutralizes the activity of megsin, it can be expected to have an effect of improving the pathological condition caused by megsin. Therefore, an antibody obtained by humanizing the monoclonal antibody of the present invention is considered to be effective for treating diseases in which mesangial cells proliferate. Usually, such a humanized antibody is preferably mixed with one or more pharmacologically acceptable carriers and provided as a pharmaceutical composition produced by a known pharmaceutical technique.
  • the monoclonal antibody of the present invention When the monoclonal antibody of the present invention is administered to humans for the purpose of treating a disease associated with megsin, a method for minimizing antigenicity against humans can also be adopted. That is, it can be carried out by converting the monoclonal antibody of the present invention into a chimeric antibody or a humanized antibody by a known method, and the monoclonal antibody of the present invention includes an antibody having reduced antigenicity against these humans. included.
  • a human chimeric antibody in which the constant regions of the H and L chains including the Fc portion of the mouse antibody were replaced with human antibodies.
  • a chimeric antibody refers to a chimeric molecule of a mouse antibody and a human antibody.
  • Human chimeric antibodies are antibodies in which the antibody variable region (V region) is derived from a non-human animal antibody and the antibody constant region (C region) is derived from a human antibody [Proc. Natl. Acad. Sci. USA, 81: 6851, 1984].
  • a mouse is immunized with a human antigen, an antibody variable region (V region) that binds to the antigen is excised from the mouse monoclonal antibody gene, and is ligated to a human-derived antibody constant region (C region) gene to produce a chimeric gene.
  • Chimeric antibodies have low antigenicity to humans, and when administered to humans, antibodies to monoclonal antibodies derived from animals other than humans are hardly elicited, and it has been reported that the half-life in blood is increased by 6 times [Proc Natl. Acad. Sci. USA, 86: 4220, 1989].
  • variable region of an antibody is defined as a region necessary for binding of an antibody to an antigen. Therefore, not only antibody fragments such as Fab that can be obtained by enzymatic digestion, but also antibody fragments that can be obtained by isolating DNA encoding the antigen-binding region and expressing it in an appropriate host vector system Included in the variable region. Furthermore, the antibody variable region of the present invention includes a scFv in which the antigen-binding regions of the heavy and light chains are linked to one peptide chain.
  • transmucosal administration in addition to oral administration and intravenous administration, transmucosal administration, transdermal administration, intramuscular administration, subcutaneous administration, and rectal administration can be appropriately selected. Accordingly, it can be used as various preparations. Hereinafter, each preparation will be described, but the dosage form used in the present invention is not limited thereto, and can be used as various preparations usually used in the field of pharmaceutical preparations.
  • the oral dose of the humanized antibody is preferably in the range of 0.1xg / kg to 100xg / kg, more preferably 1 lxg / kg to 100xg / kg. ⁇ ig / kg to 10 ig / kg.
  • Dosage forms for oral administration include powders, granules, capsules, pills, tablets, elixirs, suspensions, emulsions and syrups, which can be selected as appropriate.
  • these preparations can be modified such as sustained release, stabilization, easy disintegration, difficulty in disintegration, enteric coating, and easy absorption.
  • As a dosage form when performing oral local administration There are chewing agents, sublingual agents, buccal agents, troches, ointments, patches, liquids, etc., which can be selected as appropriate.
  • these preparations can be modified such as sustained release, stabilization, easy disintegration, difficult disintegration, enteric coating, and easy absorption.
  • DDS drug delivery system
  • the DDS preparations referred to in the present specification include sustained release preparations, topically applied preparations (troches, pacal tablets, sublingual tablets, etc.), drug release control preparations, enteric preparations, gastric-soluble preparations, etc. This refers to a formulation in the optimal dosage form, taking into account availability and side effects.
  • the components of DDS basically consist of a drug, a drug release module, an encapsulation, and a treatment program.Each component has a short half-life, in which the blood concentration decreases rapidly when release is stopped. Drugs are preferred, and encapsulations that do not react with the biological tissue at the site of administration are preferred, and it is also preferred to have a treatment program that maintains the best drug concentration for a set period of time.
  • the drug release module basically has a drug reservoir, a release controller, an energy source and a release hole or release surface. It is not necessary to have all of these basic components, and the best mode can be selected by adding or deleting as appropriate.
  • Materials that can be used for DDS include polymers, cyclodextrin derivatives, lecithin, and the like.
  • Polymers include insoluble polymers (silicone, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethyl cellulose, cellulose acetate, etc.), water-soluble polymers, and polyhydric amides (polyacrylamide, Polyhydroxyethyl methyl acrylate crosslinked product, polyacrylic crosslinked product, polyvinyl alcohol, polyethylene oxide, water-soluble cellulose derivative, cross-linked poloxamer, chitin, chitosan, etc., slowly soluble polymer (ethyl cellulose, Methyl vinyl ether ⁇ maleic anhydride copolymer partial ester, etc.), gastric soluble polymer (hydroxypropyl methylcellulose, hydroxypropylcellulose, carmellose sodium, macrocrogol, polyvinylpyrrolidone, dimethylaminoeth
  • silicon, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, methyl vinyl ether-maleic anhydride copolymer partial ester can be used for drug release control, and cellulose acetate as osmotic pump material.
  • Ethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, and methylcellulose can be used as membrane materials for sustained-release preparations, and crosslinked polyacrylic can be used as an oral mucosal or ocular mucosal adhesive.
  • solvents, excipients, coatings, bases, binders, lubricants, etc. depend on the dosage form (known dosage forms such as oral dosage forms, injections, suppositories, etc.).
  • Additives such as disintegrants, solubilizers, suspending agents, thickeners, emulsifiers, stabilizers, buffers, isotonic agents, soothing agents, preservatives, flavoring agents, fragrances, coloring agents, etc. In addition, it can be manufactured.
  • the solvent include purified water, water for injection, physiological saline, laccase oil, ethanol, glycerin and the like.
  • the excipient include starches, lactose, glucose, sucrose, crystalline cellulose, calcium sulfate, calcium carbonate, talc, titanium oxide, trehalose, xylitol and the like.
  • the coating agent include sucrose, gelatin, cellulose acetate fluorate, and the above-mentioned polymers.
  • Examples of the base include petrolatum, vegetable oil, Macguchigall, an oil-in-water emulsion base, a water-in-oil emulsion base, and the like.
  • Examples of binders include starch and its derivatives, cellulose and its derivatives, Natural polymer compounds such as ratine, sodium alginate, tragacanth, and gum arabic; synthetic polymer compounds such as polyvinylpyrrolidone; dextrin; hydroxypropyl starch;
  • lubricant examples include stearic acid and salts thereof, talc, waxes, wheat starch, macrogol, hydrogenated vegetable oil, sucrose fatty acid ester, polyethylene dalicol, and the like.
  • Disintegrators include starch and its derivatives, agar, gelatin powder, sodium bicarbonate, cellulose and its derivatives, carmellose calcium, hydroxypropyl starch, carboxymethylcellulose and its salts and cross-linked products, low-substituted hydroxypropyl Cellulose and the like can be mentioned.
  • solubilizer examples include cyclodextrin, ethanol, propylene glycol, and polyethylene glycol.
  • examples of the suspending agent include gum arabic, tragacanth, sodium alginate, aluminum monostearate, citric acid, various surfactants and the like.
  • examples of the thickener include carmellonium sodium, polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, tragacanth, gum arabic, sodium alginate and the like.
  • emulsifier examples include gum arabic, cholesterol, tragacanth, methylcellulose, various surfactants, lecithin and the like.
  • stabilizer examples include sodium bisulfite, ascorbic acid, tocopherol, a chelating agent, an inert gas, a reducing substance, and the like.
  • Examples of the buffer include sodium hydrogen phosphate, sodium acetate, boric acid and the like.
  • Examples of the tonicity agent include sodium chloride, glucose and the like.
  • Examples of the soothing agent include proforce hydrochloride, lidocaine, and benzyl alcohol.
  • Preservatives include benzoic acid and its salts, paraoxybenzoic esters, Mouth lobinol, inverted soap, benzyl alcohol, phenol, tyromesal and the like.
  • the flavoring agent include sucrose, saccharin, canzo extract, sorbitol, xylitol, glycerin and the like.
  • the fragrance, spruce tincture, rose oil and the like can be mentioned.
  • the coloring agent include a water-soluble edible colorant and a lake colorant.
  • DDS preparations such as sustained-release preparations, enteric-coated preparations or controlled-release drug preparations
  • DDS preparations such as sustained-release preparations, enteric-coated preparations or controlled-release drug preparations
  • humanized antibodies may be inactivated or degraded in vivo, resulting in reduced or eliminated desired effects. Therefore, by using a substance that inhibits a substance that inactivates or degrades DDS in combination with a pharmaceutical composition for treating and / or preventing mesangial proliferative glomerulonephritis, the effects of the components can be further sustained. These may be incorporated into the preparation or may be administered separately. Those skilled in the art can appropriately identify substances that inactivate or degrade DDS, select substances that inhibit DDS, combine them, or use them in combination.
  • ingredients other than those described above that are used in ordinary compositions can be used.
  • the addition amount of these ingredients should be a normal amount within a range not to impair the effects of the present invention. Can be.
  • monoclonal antibodies of the present invention include immunostaining, such as tissue or cell staining, immunoprecipitation, immunoblot, immunoassay, such as competitive or noncompetitive immunoassay, radioimmunoassay, ELISA, latex agglutination, protein It can also be used for purification, affinity columns and the like.
  • immunoassay includes all methods using immunological reactions such as immunohistological examination, immunoblot, and immunoprecipitation.
  • the monoclonal antibody of the present invention can be used as a labeled antibody for use in Immunoassay.
  • Enzymes, enzyme substrates, coenzymes, enzyme precursors, apoenzymes, fluorescent substances, dye substances, chemiluminescent compounds, luminescent substances, chromogenic substances Substances, magnetic substances, metal particles, radioactive substances, etc. can be used.
  • a reaction between a thiol group and a maleimide group a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, and the like can be used.
  • any form of a solution such as a colloid solution or a non-fluid sample
  • a specimen or a sample can be used as a specimen or a sample.
  • biological samples specifically, blood, plasma, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, urine, other body fluids, cell cultures, tissue cultures, tissue homogenates, biopsy samples, cells Tissue, brain tissue, brain-derived cell line, nerve cell line, nerve-derived cell line, mammary gland-derived cell line, mammary gland tissue, ovary-derived cell line, ovarian tissue, cancer cell line, cancer tissue and the like.
  • the present invention also provides such a hybridoma cell line, an immunoassay and a test kit. Further, the present invention provides a monoclonal antibody that specifically recognizes megsin, an immunoassay for detecting and quantifying megsin characterized by using the antibody, and a test kit for performing the imnoassay. You.
  • the monoclonal antibody obtained by the present invention has high specificity for megsin, and is very useful for detection and quantification of megsin.
  • FIG. 1 is a schematic diagram showing the structural difference of a reactive loop region, which is the active site of serpin, in three states.
  • FIG. 2 is a photograph of a stained image showing the result of Western blotting using the monoclonal antibody MS18a of the present invention.
  • FIG. 3 shows the neutralizing activity of the monoclonal antibody MS18a of the present invention against the inhibitory activity of the megsin protein on the plasmin enzyme activity.
  • the vertical axis represents the percentage (%) of enzyme activity neutralized by the monoclonal antibody
  • the horizontal axis represents the type of monoclonal antibody.
  • the mixing ratio of megsin is the mixing ratio of megsin.
  • FIG. 4 shows the effect of the reactive loop region peptide of megsin on the neutralizing activity of the monoclonal antibody MS18a.
  • the vertical axis indicates the percentage (%) of the enzyme activity neutralized by the monoclonal antibody
  • the horizontal axis indicates the type of the synthetic peptide used for absorption of the monoclonal antibody.
  • FIG. 5 shows the reactivity of the monoclonal antibody MS18a with PAI-1.
  • (Garden) is t_PA only
  • (Hata) is t-PA + PAI-1 + PAI-1 antibody
  • ( ⁇ ) is PA + PAI-1
  • ( ⁇ ) is t-PA + PAI-1 + monoclonal antibody MS18a
  • the change over time (0D) of the absorbance of the sample is shown.
  • the eluate was concentrated by centrifugation using Centricon lO (Millipore) (3, OOOg), and buffer exchanged with Dulbecco's PBS (-) buffer (Nissui Pharmaceutical). Centrifugal concentration and buffer exchange were repeated three times to obtain purified recombinant megsin. All operations were performed at 4 ° C using AKTAexplorer 10s (manufactured by Amersham's Biosciences) as a chromatography apparatus.
  • mice Three 8-week-old female Balb / c mice were mixed with Freund's complete adjuvant (manufactured by DIFC0) at a ratio of 1: 1 and emulsified with three immunized antigen solutions to give megsin protein of approximately 50 ig / mouse. It was injected subcutaneously. Thereafter, approximately every two weeks, the antigen solution for immunization was mixed with Freund's incomplete adjuvant (manufactured by DIFC0) at 1: 1 and emulsified to prepare a megsin protein of about 50 xg / animal. A booster immunization was performed by subcutaneous injection.
  • Freund's complete adjuvant manufactured by DIFC0
  • mice with high antibody titers were mixed 1: 1 with Freund's incomplete adjuvant (DIFC0) and emulsified to give megsin protein. It was prepared at 50 g / animal and boosted by subcutaneous injection. Three days later, spleen cells of the immunized mouse were used for cell fusion.
  • DIFC0 Freund's incomplete adjuvant
  • the protein solution of the megsin protein prepared in the same manner as the immunizing antigen was adjusted to lig / mL with PBS, and adsorbed to the ELISA plate at 100 / L / well for 4T: overnight. After washing three times with a washing solution (PBS containing 0,05% tween20 (manufactured by Wako Pure Chemical Industries, Ltd.)), blocking was performed with Block Ace (manufactured by Snow Brand Milk Products) diluted 4 times with PBS. After washing, the serum obtained in [1] was diluted 5000-fold with an antibody diluent (PBS containing Block Ace diluted 10-fold), added at 100 iL / well, and reacted at room temperature for 2 hours.
  • PBS containing 0,05% tween20 manufactured by Wako Pure Chemical Industries, Ltd.
  • Block Ace manufactured by Snow Brand Milk Products
  • a 5000-fold diluted peroxidase-labeled mouse IgG antibody (manufactured by Chemicon) was added at a rate of 100 L / well, and reacted at room temperature for 2 hours.
  • the plate was washed 5 times with the washing solution, and the substrate solution was added at 100 L / well. After reacting with the substrate solution for 30 minutes, the reaction was stopped by adding 100 zL / well 2N sulfuric acid, and the absorbance at 490 nm was measured.
  • the spleen was excised from three mice in which an increase in the antibody titer against the megsin protein was observed as a result of the ELISA method, and spleen cells were prepared by a conventional method.
  • the parent strain at the time of cell fusion was selected in advance in a medium containing 20 g / ml of 8-azaguanine and confirmed to be a hypoxanthine / guanine / phosphoribosyltransferase (HGPRT) deficient strain.
  • HGPRT hypoxanthine / guanine / phosphoribosyltransferase
  • One SP2 cell line was used. SP2 cells 2 x 10 combined 7 splenocytes IX 10 8 pieces of polyethylene glycol 4000; using (PEG 4000 manufactured by Merck) as a cell fusion promoter, were performed cell fusion according to a conventional method.
  • the cells after fusion are suspended in a medium (HAT medium) containing hypoxanthine, aminopterin and thymidine in Escron medium (manufactured by Sanko Junyaku) at a concentration of 3.0 ⁇ 10 8 cells / mL in spleen cells. 100 L / well was dispensed into a well microphone opening plate (manufactured by Corning). The fused cells 3-5 were incubated with the medium was replaced by half the daily in C0 2 incubator (37 ° C, 5 C0 2 ). Only the hybridomas that can be cultured in the HAT medium were selectively cultured. e. Hybrid screening
  • the isotype of the monoclonal antibody produced by the hybridoma MS18a cell line was IgGl for the H chain and ⁇ for the L chain.
  • the obtained supernatant was purified using an affinity column Sepharose Protein in A (manufactured by Amersham Bioscience) to purify a monoclonal antibody (MS18a).
  • MS18a monoclonal antibody
  • the absorbance of this antibody solution at 260, 280, and 320 nm was measured, and the antibody concentration was measured by the Welbulg-Christian method.
  • the membrane was reacted with the monoclonal antibody MS18a diluted with an antibody diluent for 4 times. Then, an alkaline phosphatase-labeled mouse IgG antibody was added, and the mixture was reacted at room temperature for 1 hour, and then colored with an NBT-BCIP solution. The result is shown in figure 2.
  • Lane 1 shows plasmin, one of the ligands of megsin protein, lane 2 shows megsin protein, and lane 3 shows a mixture of megsin protein and plasmin.
  • (a) shows the result of the reaction with the monoclonal antibody having no neutralizing activity
  • (b) shows the result of the reaction with the monoclonal antibody MS18a of the present invention.
  • Example 2 Neutralizing Activity of Megsin Protein Against Plasmin Enzyme Activity Inhibitory Effect of Megsin on the Reaction of Megsin with Plasmin, One of Megsin Ligand, Neutralizing activity against plasmin enzyme activity inhibitory activity was examined. Affects megsin activity for comparison , A monoclonal antibody that was confirmed not to give the same was used.
  • the protein solution of the megsin protein prepared in the same manner as the immunizing antigen was adjusted to 0.5 mg / mL with Buffer P (0.1 M Tris-HCl buffer ( ⁇ ) containing 0.05% tween 20), and then adjusted to 96 mg / mL.
  • Buffer P 0.1 M Tris-HCl buffer ( ⁇ ) containing 0.05% tween 20
  • NUNC Pell Black microplate
  • Example 1 to 2 1, 1: 1 and 1: 2 (molar ratio).
  • PBS 0.1 M Tris-HCl buffer
  • plasmin manufactured by SIGMA
  • SIGMA SIGMA
  • a substrate solution (buf fer P containing 0.526 mM Boc_Va Leu-Lys-MCA (manufactured by Peptide Research Laboratories) was added at 95 L / well, and allowed to react at room temperature for 1 hour. Then, the fluorescence intensity (Ex 380 nm, Em 460 nm) was measured.
  • the fluorescence intensity of plasmin only, plasmin and megsin, plasmin and MS18a, and plasmin, megsin and MS18a minus the fluorescence intensity of the substrate solution only (blank) are FP, FPMeg, FP18, and FPMegl8, respectively.
  • Neutralizing activity was determined as follows. As shown in FIG. 3, the monoclonal antibody MS18a neutralized the inhibitory effect of the megsin protein on the plasmin enzyme activity.
  • a protein solution of the megsin protein prepared in the same manner as the immunizing antigen was adjusted to 1 g / mL with PBS, and adsorbed to an ELISA plate at 100 ⁇ 17 ⁇ for 4 overnight. After washing three times with a washing solution (0.02% Tween20 / PBS), blocking was performed with Block Ace diluted 4 times with PBS. Separately, a peptide of 9 to 17 residues was synthesized by a conventional method, centering on the cleavage site ( ⁇ - ⁇ ') of the reactive loop region (P17-P5': EEGTEATAATGSNIVEKQLPQS / SEQ ID NO: 1) of the megsin protein ( table 1 ) .
  • Disodium p-nitrophenylphosphate SIGMA 104 phosphatase substrate tablets
  • a substrate reaction solution 9.6% diethanolamine buffer (pH 9.7) containing 0.5 magnesium chloride
  • the substrate was dissolved to prepare a substrate solution.
  • the plate was washed five times with the washing solution, and the substrate solution was added at 100 L / well. After reacting with the substrate solution for 30 minutes, 100 ⁇ L of 3N NaOH was added to stop the reaction, and the absorbance at 405 nm was measured. Controls consisted of no peptide addition and the addition of an irrelevant peptide to the peptide. High absorbance indicates that the antibody has not reacted with the peptide. Table 1 shows the results.
  • M107, M108, MH1, and M112 of the M107 to M112 peptides that showed a large change in absorbance were adjusted to lmg / mL with PBS, and the 96-Elpla microplate ( NUNC) (1.5 L / well) and mixed with the monoclonal antibody MS18a (lmg / mL, 1.5 L / well) purified with protein A. After reacting at room temperature for 1 hour, the protein solution of the purified megsin protein was adjusted to 0.5 mg / mL with buffer P, and 1 L / well was added and mixed.
  • the fluorescence intensity of plasmin only, plasmin + megsin, plasmin + monoclonal antibody MS18a, and plasmin + megsin + monoclonal antibody MS18a minus the fluorescence intensity of the substrate solution only (blank) was FP, FPMeg
  • the neutralizing activity was determined in the same manner as in Example 2 with FP18 and FPMegl8 (FIG. 4). From Table 1 and FIG. 4, the neutralization activity decreased in the reaction with M107 and M112. This indicates that the antibody did not react with megsin but reacted with the peptide.
  • the epitope common to M107 and M112 is NIVEKQ (SEQ ID NO: 4). Therefore, the peptide NIVEKQ
  • PAI-1 (Molecular) adjusted to 0.2 mg / mL with 0.2 M Tris-HCl buffer (pH 8)
  • Escherichia coli expressed fragmented megsin shown below was prepared.
  • Fragment amino acid sequence 4-9: 267-321 Total length: 1-380 5_10: 322-380
  • the fragmented megsin prepared in (1) was electrophoresed and subjected to Western plot to confirm bands reacting with the monoclonal antibodies MS44b and 4F3.
  • the results are shown in Table 2.
  • ⁇ indicates that a band reacted with the monoclonal antibody was observed Indicates that X was not observed.
  • Peptides were synthesized based on the data of fragmented Escherichia coli megsin, immobilized on a plate (Kovalink NH module, manufactured by Nunc), and ELISA was performed as follows.
  • the synthetic peptide was immobilized on a plate by the amine coupling method. After washing twice with the washing solution, add Block Ace diluted 4 times with PBS to block. Add the diluted antibody (100 / iL / well) to the plate and incubate at room temperature for 2 hours. After washing 4 times with the washing solution, 13 ⁇ 43 ⁇ 4 is reduced to 100 1 1 The reaction was carried out at room temperature for 30 minutes, and the absorbance at 450 nm was measured. The results are shown in Table 3.
  • the peptide was immobilized on the CM5 chip by the amine coupling method, and the reactivity with the antibody was measured by biascore. The results are shown in Table 5.
  • the epitope was present in the range of 245-260 for the monoclonal antibody 4F3 and in the range of 305 for the monoclonal antibody MS44b. While the monoclonal antibody of the present invention having a neutralizing activity recognizes the amino acid sequence of SEQ ID NO: 4 (corresponding to 343-348 in the amino acid sequence of megsin) as an epitope, it has a neutralizing activity. Monoclonal antibodies that do not have a sequence of SEQ ID NO: 4 or less, such as 245-260 or 29-305 It was confirmed that the outer area was recognized as an epitope. Industrial applicability
  • a monoclonal antibody that neutralizes the activity of megsin was provided. Further, according to the present invention, it was confirmed that an antibody that binds to the reactive loop region of megsin can neutralize the activity of megsin.
  • Megsin is a molecule that causes mesangial matrix expansion, glomerular cell proliferation, and immune complex deposition in the glomerulus. These lesions are characteristic of human mesangial proliferative glomerulonephritis. Expansion of the mesangial matrix is an important lesion in many renal disorders, including mesangial proliferative nephritis.
  • a monoclonal antibody that neutralizes the activity of megsin is useful in treating diseases involving expansion of the mesangial substrate.
  • the present invention also relates to a method for treating and / or preventing mesangam-proliferative glomerulonephritis, comprising as an active ingredient a monoclonal antibody that neutralizes the activity of megsin, a variable region thereof, or an antibody variable region including a complementarity-determining region thereof.
  • a composition for is provided.
  • megsin causes mesangial proliferative glomerulonephritis.
  • antibodies that neutralize megsin are useful for treating or preventing mesangial proliferative glomerulonephritis.

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Abstract

L'invention concerne un anticorps monoclonal neutralisant l'activité de la megsine. Cet anticorps monoclonal neutralise l'effet inhibiteur de la protéase de la megsine, par exemple, en se liant au domaine de boucle réactive de la megsine. En outre, l'invention concerne des compositions permettant de traiter et/ou de prévenir une néphrite glomérulaire proliférative mésangiale, lesquelles compositions contenant l'anticorps monoclonal mentionné ci-dessus ou son domaine variable, en tant que principe actif.
PCT/JP2003/004459 2002-04-11 2003-04-08 Anticorps monoclonal neutralisant la megsine WO2003084998A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006093128A1 (fr) * 2005-02-28 2006-09-08 Tokai University Educational System Anticorps monoclonal anti-megsine
WO2006121047A1 (fr) * 2005-05-09 2006-11-16 Tokai University Educational System Procede de determination d’une megsine dans un echantillon biologique
JP2008501308A (ja) * 2004-02-20 2008-01-24 ルドウィグ インスティテュート フォー キャンサー リサーチ Ｅｇｆレセプターエピトープペプチドおよびその使用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIJNENS A.P. ET AL.: "The distal hinge of the reactive site loop and its proximity: a target to modulate plasminogen activator inhibitor-1 activity", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 48, 2001, pages 44912 - 44918, XP002217300 *
TOSHIO MIYATA ET AL.: "A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 102, 1998, pages 828 - 836, XP002969523 *
TOSHIO MIYATA ET AL.: "Overexpression of the serpin megsin induces progressive mesangial cell proliferation and expansion", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 109, no. 5, 2002, pages 585 - 593, XP002969524 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008501308A (ja) * 2004-02-20 2008-01-24 ルドウィグ インスティテュート フォー キャンサー リサーチ Ｅｇｆレセプターエピトープペプチドおよびその使用
JP2012102109A (ja) * 2004-02-20 2012-05-31 Ludwig Inst For Cancer Research Ｅｇｆレセプターエピトープペプチドおよびその使用
WO2006093128A1 (fr) * 2005-02-28 2006-09-08 Tokai University Educational System Anticorps monoclonal anti-megsine
WO2006121047A1 (fr) * 2005-05-09 2006-11-16 Tokai University Educational System Procede de determination d’une megsine dans un echantillon biologique

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