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WO1997049427A1 - Medicament permettant d'attenuer les problemes renaux - Google Patents

Medicament permettant d'attenuer les problemes renaux Download PDF

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Publication number
WO1997049427A1
WO1997049427A1 PCT/JP1997/002241 JP9702241W WO9749427A1 WO 1997049427 A1 WO1997049427 A1 WO 1997049427A1 JP 9702241 W JP9702241 W JP 9702241W WO 9749427 A1 WO9749427 A1 WO 9749427A1
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WIPO (PCT)
Prior art keywords
phospholipase
ser
type
antibody
thr
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PCT/JP1997/002241
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English (en)
Japanese (ja)
Inventor
Yasushi Kawauchi
Jun Takasaki
Kazumi Hayashi
Yasuhiko Masuho
Original Assignee
Yamanouchi Pharmaceutical Co., Ltd.
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Filing date
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Application filed by Yamanouchi Pharmaceutical Co., Ltd. filed Critical Yamanouchi Pharmaceutical Co., Ltd.
Priority to AU32761/97A priority Critical patent/AU3276197A/en
Publication of WO1997049427A1 publication Critical patent/WO1997049427A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to an agent for improving kidney injury, and more particularly to an agent for preventing, modifying, or treating a disorder caused by administration of a gold compound.
  • I-metallized compounds are anti-Mi ⁇ drugs and are widely used for urinary / genital tumors in the polar region as well as in the iH: region.
  • bladder cancer ovarian cancer, cervical cancer, esophageal cancer, cancer and other panpan.
  • the bone marrow gap 'there are side effects the river, such as Utsuwasuki 1 ⁇ 2, especially ⁇ ' hearing is low / 11 ri:! Order to occur in 1 to ⁇ mn Because of this, throw ⁇ . Is restricted.
  • Cisplatin is filtered from the glomerulus and is secreted from the proximal fibrils and excreted in the urine, so that cisbratin at the skin ft depth; Renal damage is seen in the proximal renal fine of ⁇ , in extraordinary I un-h; ⁇ cause fine ⁇ necrosis of the state (Shigeru Kimura ⁇ ::., Et al., 1 ⁇ of Cis-Diaminedichloroplatinum (1 1); of electron microscopy , E1 Urinary Society 76: 1439-1453, 1985).
  • 24-hour continuous infusion Jac obs, C. et al., 24-hour infusion ⁇ cis-platinum in head and neck cancer.
  • the suppression of renal injury by cisplatin is defined as F1 ⁇ vasodilator, adenosine A1 receptor antagonist, radical scavenger, lysolime membrane stabilization ⁇ (Deegan, PM et al. Renal Failure 17, pll7-123 (1995), Nagashima, K. et al. Jpn. J. Pharmacol. 67, p349-357 (1995), Mc Ginness, JE et al. Physiol. Chem. Physics. 10, p267-277 (1978) Lab. Invest. 55, p557-563 (1986), Kobayashi; Akira, et al., Japan Obstetrics and Gynecology Association 41. ⁇ 683-687 (1989)), etc. ⁇ ', but not to clinical Okawa. Under these circumstances, the use of cisplatin ⁇ ⁇ ) is a drug that can be used as a drug.
  • JJ Phospholipase
  • Phospholipase is known as an element that hydrolyzes the ester bond at the C2 position of 1,2-diacylphosphoglyceride, which is a component of the upper resting component.
  • This enzyme is found in the extreme organs and cells of ⁇ ⁇ ⁇ ⁇ , and not only regulates the metabolism and metabolism of biological membrane phospholipids, but also has the role of arachidonic acid cascade as a yeast: It is known that the products of the process, such as prostaglandin, leukotriene, tromboxane, and PAF, have various properties.
  • Phospholipase ⁇ 2 contains ⁇ , ⁇ ⁇ ! The intracellular ⁇ is known (II IIRa ⁇ Clinic, 1994 Special Issue, 202-206). Even in this block, the type 11 phospholipase ⁇ 2 is induced into the exudate block of the inflamed site along with the inflammation reaction, and many ⁇ are released into the blood. In addition, there are rare reports suggesting that this enzyme is a part of the cause of the disease in some inflammatory diseases.
  • Kikuchi- Yano shita discovered that distribution I 1 to the model of ⁇ Blood heart disease in rats, a model of myocardial infarction in correspondence with the progress of organ ⁇ harm the activity of this enzyme is elevated; to (K i kuc hi -Ya noshita, R. et al., J. Biochemistry, 114: 33-38, 1993)
  • Monoclonal antibody that releases II phospholipase bound to the cell crotch which is a monoclonal antibody, or a part thereof, is used to test the antimicrobial activity.
  • Pharmaceutical composition which is a modification of ⁇ ⁇ by administration of Guangqi ⁇ ⁇ ⁇ as an active ingredient
  • Phospholipase ⁇ 2 is human! The composition according to any one of (1) to (3),
  • the monoclonal antibody has a function of releasing human native type II phospholipase A2 bound to the cell membrane, and the monkey f1: l native type II phospholipase A2 bound to the cell membrane and / or mouse-derived type 11 phospholipase A2 (2) the medical composition according to (2), wherein
  • Monoclonal antibody is one of the types II phospholipase A2 derived from human, and the monkey I II's phospholipase A2 and / or mouse Yamaki 11 11 '! (3)
  • the pharmaceutical composition according to (3) which is a monoclonal stake rest that is capable of acting and capable of releasing 11 ⁇ phospholipase ⁇ 2 that has been linked to cell detachment.
  • Antibodies refers to ⁇ - ⁇ ⁇ ⁇ that are / -stimulated by the immune response, are '- ⁇ in the rest of the day, and have the activity to specifically bind to the immune j (antigen).
  • a “monoclonal antibody” is an antibody that is produced by a single-clone pile rest cell (Biochemical Dictionary 2nd ed., 1359 ⁇ , Tokyo Chemical Co., Ltd. (1990)).
  • ⁇ White matter is the 'L: essential component of an organism, consisting of a polypeptide chain in which approximately 20 L-amino acids (including glycine) are linked by peptide bonds. ; : 'Dictionary 2nd edition, 810 shellfish, ⁇ ⁇ Chemical Doujin (1990)) ⁇
  • Peptide means two or more amino acids joined by a peptide ⁇ It means that the number of amino acids is about 10 or less is called lipopeptide, and the one with less than 1 is called polypeptide (Biogenesis ⁇ Dictionary 2nd edition, 1202 ⁇ ⁇ Kagaku Doujin (1990)).
  • Epitope means the structure of the i-position that is present in the antigen component f-10 and identified by the antibody. ( ⁇ Chemical Dictionary 2nd ed., 195 ⁇ , Tsukakyokadoujin (1990); ⁇ , Bio i Shinkawa Dictionary 4th Edition, 93, Saito Biotech (1995)).
  • revision j refers to an agent that reduces the harm of the A metallized pedestal and reduces the damage.
  • the pile rest of the Hjj, antibodies as possible out to inhibit the ' ⁇ ⁇ ⁇ of II phospholipase A2 Kuikyu is preferably dry.
  • ⁇ ⁇ : from the things ⁇ rest to W.1 is the river Iruko Toga ⁇ 1 ⁇ a, ⁇ No 'A monoclonal antibody with' reaction ' would be suitable. It is also known that phospholipase A2fi ':?
  • Antibodies that release phospholipase A2 enzyme from cell membranes Is used.
  • Specific antibodies include, for example, the monoclonal antibody described in International Publication No. ⁇ 96 / 20959, and the monoclonal antibody 12 ⁇ 5, 10.1 or 1.4 is preferred. Is more preferred.
  • hybridomas 12H5, 1.4, and 10.1 These monochrome piles have an nj ability to pick up and remove hybrids that have these / rests, namely, the hybridomas 12H5, 1.4, and 10.1.
  • hybridomas 12H5, 1.4, and 10.1 These hybridomas were obtained by increasing the immunity and increasing the immunity Kfj'ij, and spleen cells of BALB / c mice sensitized with 11-phospholipase A2 in healthy volunteers and Pffix cells of mouse P3x63Ag8 / U 1 (P3U1) can be fused by a conventional method, for example, the cell fusion fY method of Kayla and Milstein, and j: is effective (International Publication No. 96/20959 ⁇ ' ⁇ ⁇ / j Details).
  • the mouse hybridomas 12H5, 1.4, and 10.1 obtained by the present inventors have been deposited as follows.
  • Dulbecco's modified minimum essential medium As a medium for culturing the hybridoma, for example, Dulbecco's modified minimum essential medium (Dalbecco's modified minimum essential medium; hereafter abbreviated as “DMEMj”) is used.
  • DMEMj Dulbecco's modified minimum essential medium
  • a medium containing glutamine, glucose, sodium pyruvate, 2-mercaptoethanol, and stake organisms for example, penicillin 0, streptomycin, genyumycin, etc. is used.
  • Hybridoma cultures are usually 5% at 37 ° C in medium 11 '-carbon oxide, 95% empty In the abdominal cavity of BALB / c mice pre-treated with 2,6,10,14-tetramethylpentyldecane (e.g., Aldrich i, Pristane) for 2-4 days in the gas phase
  • the purification is carried out in about 10 to 20 ⁇ g to produce antibodies that can be purified.
  • the antibody used in the liquid assay may be a part thereof, or may be a part retaining the action of inhibiting II phospholipase A2 activity.
  • Pile rest is digested with a protease such as pepsin by a conventional method, and is isolated and purified by a conventional method for decoupling I'l protein. ) You can get 2 .
  • the H chain and H promotion of the pile body and the H / A are converted to U (: I) by the dialkylation of the disulfide bond connecting H'i and L by the flow of dithiothreitol and odoacetamide.
  • Hl and L ⁇ ' ⁇ can be ⁇ littered 'by the J bond alone, and the ⁇ alkyl rest can be achieved (“Useful immunity test method I: Scientific, 1984”) j See 39 shellfish).
  • Monoclonal antibody lysate that is introduced in the water is used to inject proteins or peptides that contain the components of the stake.
  • Examples of such monoclonal suspensions include: (1) an antibody consisting of a single amino acid sequence derived from an animal such as a mouse only in the i and J variable regions, and a normal region consisting of the amino acid sequence of human wildlife.
  • a so-called “chimeric antibody” (2) only the complementarity determining region (or super- one- variable region: abbreviated as "CM_i” (abbreviated as “omplementarity-determining region”))
  • the remaining region is composed of amino acid sequences, and the other region is a stake rest consisting of the amino acid K sequence of human wildlife, that is, a so-called "humanized an tibody”.
  • “Chimeric pile rest” or “humanized antibody” means, for example,
  • SEQ ID NOs: 2, 4, and 19 are shown in any of 21.
  • the amino acid sequence listed above is CDR part (SEQ ID NO: 5, 6, 7, 8, 9, 10, 22, 23) , 24, 25, 26
  • a DNA containing a base sequence encoding 27) is also preferably used.
  • the chimeric antibody according to the present invention which encodes humanized anti-bacterial activity, - for inhibiting the activity, it is also possible to have ffl the IgG 4 ⁇ Ko as a framework portion (J.Exp.Med.166,1351-1361 (1987)).
  • the gene encoding the antibody can be isolated from the hybridoma producing the antibody of the present invention by a known method (Cancer Research, 47, 999-1005 (1987): Proc. Natl. Acad. Sci. .84, 2936-2940 (1987)). For example, it is possible to ⁇ ) separate from the aforementioned mouse hybridomas 12H5, 1.4, 10.1 by a known method.
  • the protein or peptide comprising the monoclonal pile of the present invention or a part thereof may be incorporated into the expression vector by incorporating the ordinal portion or a part of the gene encoding the antibody of the present gf! Ij, Large m ⁇ , fermented fii or ⁇ of course to the cell! It is the "J ability" that can also be obtained by letting them do it.
  • the amino acid exchange and the deletion of the antibody or a part thereof introduced in the present invention can guide the change of import.
  • the introduction of an amino acid alteration can be performed by introducing an alteration such as base substitution, ablation or insertion into a DNA encoding the amino acid.
  • the conversion of DNA can be conducted by a known method (Gillamn et al., Gene, 8, 81-97 (1979): Roberts et al. 'Nature, 328, 731-734 ( 1987)).
  • a force method based on the phage display method (Annu. Rev. Immunol., 12,433-455 (1994)).
  • Monoclonal antibodies 12H5 and 10.1 ligase can also bind to the epitope linked by 1.4.
  • Those skilled in the art can easily identify the peptide to which a specific monoclonal antibody binds by using a conventional method (see “Monoclonal Experiment J. (See Tokyo Kagaku Doujin (1989).)
  • various monoclonal antibodies that bind to the epitope can be obtained by immunization using the synthesized epitope or the like. Yes (E. Cordelia et al., Proc. Natl. Acad. Sci. USA, 90, 10290-102 94 (1993)).
  • all or a part of the antibody clones directed against a part thereof may be subjected to an ELISA method using an epitope to screen a specific monochrome-null pile. It is possible.
  • the agent for improving renal impairment of the present invention is determined depending on the case in consideration of the administration route, disease symptoms, age and sex of the administration subject, and the like.
  • the renal stimulant-improving agent of the present invention is particularly effective when an antibody is used as an active ingredient, non-oral injection, that is, subcutaneous, intramuscular or intravenous injection.
  • I-streams usually consist of a solution of protein or peptide in a water-repellent, preferably aqueous, solution. Each aqueous solution can be used 4 times, for example, water, buffer, 0.4% saline, (1.3% glycine, 5% glucose, human albumin solution. These solutions are free.
  • Auxiliary materials that are necessary for approaching Ui! Conditions such as chemicals, etc., are used as vegetative materials, for example, sodium acetate, sodium chloride, chlorination power It can produce lithium, calcium chloride, sodium lactate, sodium citrate I, and lium ji.
  • Well-known ridges have the ability to perform well-known techniques, such as, for example, Remington's Pharmaceutical Science, l5J; k, Mack Publ.
  • 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 3 ⁇ 4 'It can be used by dissolving it in a suitable solvent, and can be used in freeze-drying and ffi-living.
  • the administration method is as follows: [Before gold compound administration, at the same time as I gold compound injection, or after gold compound injection 'J. 3 ⁇ 4Can submit drug accusations.
  • the dose m of the monochromatic pile rest that is used in the present development is preferably 0.1 mg / kg to 500 mg / kg, and is usually injected into the pulse.
  • ⁇ ' is a joint action machine i ⁇ , (3 ⁇ 4found and ,, 22, ll, p57 (l 988), Physician's desk reference, BOedition p705 (1996))
  • Compounds with anti-inflammatory properties such as cisplatin, carboplatin, nedaplatin, ticoblatin, or ⁇ cancer chemotherapy '' (p27, 1990-1991, Chugai ' 254-5, DWA2114R, NK121 and the like described above.
  • cisplatin or carboplatin Simple theory of drawing
  • Figure 2 shows the results of the ELISA for 10.1 and 1.4 for 'li liU Koitayama ⁇ phospholipase A2 ⁇ '.
  • [1]] 4 shows the inhibition of 12H5, 10.1 and 1.4 against rat II phospholipase A2.
  • J6 shows an additive effect on each of the polar platelet Yamato phospholipases A2? V, 'I: of 10.1.
  • 1-18 indicates the action of releasing phosphorivase-2 bound to ⁇ -3A cells at 12 ⁇ 5, 1.1 and 1.4.
  • This section describes the method for anchored PCR cloning of the 1.4 and 12H5 positions' ⁇ and Lii'J: region cDNA.
  • M10 is the cDNA sequence and translated amino acid sequence of the uj domain of L Is shown. In addition, it is predicted to correspond to the CDR sequence (the row is labeled with a linear force.
  • FIG. 11 shows the cDNA sequence and translated amino acid sequence of the variable region of the H chain of antibody 1.4. The sequences predicted to correspond to the CDR sequences are underlined
  • Figure 12 shows the method for PCR cloning of the H and L chain variable regions cMA in 10.]
  • Figure 13 shows the piles 1 shows the cDNA sequence and translated amino acid sequence of the variable region of the L chain of 12H5, and the sequence corresponding to the CDR sequence is underlined.
  • I 14 shows the cDNA sequence and translated amino acid sequence of the variable region of the heavy chain of antibody 12H5. Note that the sequence that is considered to correspond to the CDR sequence is marked with a dotted line. l3 ⁇ 4l 15 shows phospholipase A2 i.: in mouse 4 ′ after cisplatin injection.
  • f3 ⁇ 4l 16 shows the mouse “I'y-GTPgu properties” after administration of cisplatin.
  • FIG. 17 shows mouse BUN i at 4 4! I after administration of cisplatin.
  • Moto monochrome one monoclonal antibody is ⁇ is by immune a Rikonbinan Bokuhi Bok II phospholipase A2, but recombinant human WINCH 1 1 3 ⁇ 4 Hosuhoripa its tone ⁇ all Japanese ⁇ 7 5- 192167 ⁇ Publication Ichize ⁇ 2 Performed according to the method described above.
  • BALB / c strain male mice the (seismic & 6 weeks old at ⁇ Hajimeji), ginseng ⁇ Example la) ⁇ animals the convenor emissions Bok human type II phospholipase A2 or purified, 1 'I Ri 20 ⁇ G, 0 , 1 ml of ⁇ ⁇ .
  • Saline solution was mixed with Freund's complete adjuvant, emulsified, and injected intracrotally (primary immunization). Afterwards, 2-3 ⁇
  • nj phospholipase ⁇ 2 Was mixed with Freund's complete adjuvant of the same volume and emulsified, and a booth was thrown.
  • phospholipase A2 Twenty-four days after the seventh immunization, 20 / g of phospholipase A2 per animal was dissolved in 0.2 ml of physiological saline and administered intraperitoneally (final immunization). Three days after the final immunization, splenic armpit cells were collected from 1 P mice and suspended in DMEM-ground.
  • DMEMlOml kept at 37 ° C was dropped to terminate the cell lysis reaction.
  • the reaction mixture was centrifuged, after removing the h3 ⁇ 4 solution, HAT medium [hypoxanthine (1x10 ' ⁇ ), Ami Noputerin (4x10- 7 ⁇ ), 10% ⁇ sheet fetal thymidine was added (1 .6x10 ⁇ ⁇ ) It was added to the residue DMEM medium] containing linf, spleen cell concentration; was set to 6XlO s cell number / ml. The suspension was dispensed into a 96-well plastic plate at a rate of 200 ⁇ 1 ( ⁇ 1.2 ⁇ 10 : 'cells) per well.
  • the medium was removed by suction, and the medium was added. Cell fusion? About 10 holes later, about half of the holes had Hypri-doma ⁇ ⁇ .
  • the antibody activity in the supernatant was measured by the method of d). Positive clones were transferred to a 48-well plastic plate and further transferred to a 24-well plastic plate and cultured in the same manner as described above except that aminopterin was eliminated. On a 24-well plastic plate culture, the antibody activity was confirmed by the method of d), and the enzyme inhibitory activity was measured by the method of e).
  • the purified recombinant human type II phospholipase A2 was dissolved in 0.05 ⁇ g / ml of 20 mM Tris-buffered saline (TBS, pH 7.4) in each well (well) of a 96-well incisor microplate. And stored at 4 ° C overnight in a humid chamber. Thereafter, the solution is discarded, and TBS containing 1% blood serum albumin (hereinafter, abbreviated as BSA) is added in 200 ⁇ 1 each, and incubated at 37 ° C for 1 hour to block an adsorbed portion of each gel. did.
  • TBS Tris-buffered saline
  • BSA blood serum albumin
  • the o High Priestess dormer culture say yes ascites say yes that was TBS (TBS-Tween) several times with a solution washing ⁇ containing 0.05% Tween 20 (Tween 20 trade name): the Seikuikyushirube ⁇ , 0.2% BSA
  • TBS-Tween BSA-TBS-Tween
  • Recombinant human type II phospholipase A2i 0.5 ⁇ and a culture supernatant or a purified pile were prepared using a 125 mM Tris-HCl buffer containing 150 mM sodium chloride, 12.5 mM calcium chloride, and 250 ⁇ g / ml BSA.
  • the solution (pH 8.0) was subjected to preincubation at room temperature for 2 times at room temperature.
  • 25 ⁇ ⁇ ⁇ 1 (10 cpm from 5 nogo) of the autoclave SN17 containing tritium-labeled oleic acid was added to the reaction solution, and the mixture was reacted at 37 ° C for 30 minutes. I let you.
  • reaction was transferred to ice, and 4N hydrochloric acid was added in 25 ⁇ 1 to give ⁇ II :. After the reaction was stopped, 25 mg / ml of 40 mg / ml BSA was added, mixed, and then released on ice for 30 minutes.
  • the supernatant was centrifuged at 15000 rpm for 2 minutes to measure the radioactivity of the supernatant.
  • the value obtained by subtracting the negative control (radioactivity of a sample not containing phospholipase II and antibody) from the obtained radioactivity was defined as the phospholipase A2 activity.
  • the Ml ',' ratio for the phospholipase A2 activity of the antibody was calculated based on the phospholipase-2 activity of the positive control (phospholipase-2 activity of a sample containing only the phospholipase-2 and not producing the antibody). It was a fraction.
  • Phospholipase A2 (1) (Phospholipase A2 and I, 1 ⁇ Phospholipase A2 i,) / (n-control phospholipase A2 activity)
  • the same volume of binding buffer was added to the gamma globulin fraction, mixed, and then mixed with the same binding buffer, and the column (gel) filled with “Protein A-Sepha rose CL4B” (Pharmacia) was used.
  • the solution was applied to a bed volume of 20 m 1) and washed with three columns of binding buffer.
  • IgG was eluted with about 3 column volumes of the output buffer contained in the key.
  • the eluted IgG was dialyzed against 20 mM Tris-buffered saline (PH7.4) and the like, and used as an antibody standard. Usually, 5 to 10 mg of IgG was obtained in 1 ml of ascites.
  • the IgG subclass was determined using Amersham's mouse monoclonal antibody, Swiping River kit. This method was based on the ELISA method and determined to be 12H5 (IgG2a), 1.4 (IgG2a), and 10.1 (IgGl).
  • the solution was passed through a sulfated cell mouth fine (Seikagaku Corporation) column (econo column; column size 20 ml; BIO-HAD) equilibrated with O.IM acetate buffer (pH 6.0). 200 ml of washing solution (Om acetate buffer (pH 6.0) containing 0.5 M sodium chloride) was passed through the column to wash thoroughly, and the eluate (0.1 M acetate buffer containing 1.5 M sodium chloride (p. H6.0)) was passed through 30 ml of the solution, and the resulting fractions were used as sulfated cell-port fine bonded I solids. Next, the sulfated cellulomin-bound fraction was separated by HPLC.
  • the column was CAPCELL PAK C18 (4.6 Wake 10 2501]; ⁇ / 1: manufactured by Dodo), and the HPLC system was LC-4A 3 ⁇ 4 ⁇ manufactured by Seiko Seisakusho), all at a flow rate of 1 ml / min.
  • the solution was passed through 0.1% h trifluoroacetic acid for 60 minutes.
  • phospholipase A2 was dissolved in a linear gradient of 0-50% acetonitrile under 0.1% trifluoroacetic acid ⁇ 1 :. Since the peak of phospholipase A2 appears at about 30% acetonitrile, this peak was collected.
  • PRP platelet 3 ⁇ 4 ⁇ plasma
  • EDTA is added to a final concentration of 2 mM
  • the cells are centrifuged at 2500 xg for 10 minutes.
  • the cells were suspended in 30 mM Tris-HCl buffer (pH 7.4) containing 120 mM sodium chloride and 2 ⁇ EDTA, and centrifuged at 2500 xg for 10 minutes.
  • the dialysis operation was performed on 5 OrnMff acid buffer (pH 4.5) containing 200 mM sodium chloride. After the dialysis, the cell lysate was centrifuged at 15,000 xg for 40 minutes at 4 ° C, and the ⁇ ⁇ ' ⁇ ' was collected to obtain platelet Yamagi phospholipase II.
  • the IgG preparation was adjusted to 10 ⁇ g / ml with BSA-TBS-Tween to prepare a three-fold dilution series.
  • An ELISA was performed according to Reference Example Id), and the interaction with recombinant human phospholipase A2 and rat phospholipase A2 was performed for tl.
  • ELISA against rat II phospholipase A2 was carried out by replacing reference recombinant Id) glue-combinant human phospholipase A2 with rat II type phospholipase A2.
  • FIG. 3 shows the inhibition ratio of each antibody against the recombinant human type II phospholipase A2 activity by taking the concentration of the antibody upon preincubation of the antibody preparation with phospholipase A2 on the horizontal axis. 12H5 and 1.4 almost completely fluffed phospholipase A2 at a concentration of 10 ⁇ g / ml. On the other hand, 10.1 showed an 80% inhibition at a concentration of 3 ⁇ g / ml).
  • Figure 7 shows. 12 ⁇ 5, 1.4 and 10.1 showed that phospholipase 2 activity from human liil platelet ill was recombined with recombinant human phospholipase 2
  • I'm c'i! The rest had the ability to identify i of natural human 11 phospholipase A2.
  • the antibody of the invention was a PU from phospholipase A2 ⁇ 'i: from rhesus monkeys and mice. In addition, the-part of the phospholipase A2 activity of dog fill Koitayama was determined.
  • I0D0GEN manufactured by Pierce Co., Ltd.
  • 100 ⁇ M lOOmM Tris-HCl buffer ( ⁇ 7.4) containing 30 ⁇ g of recombinant human type II phospholipase A2 was added to the test.
  • 6 ⁇ 1 of 100 mCi / ml of Na 2 ('I (ICN ⁇ )
  • the reaction solution was taken out from the glass test tube to remove the ⁇ . . Na 1 2 5 1 of reaction was removed by gel filtration.
  • the radioactivity of the culture solution was measured when the purified antibody preparation of each degree was added to the cells to which phospholipase A2 had been bound and incubated.
  • the horizontal axis shows the antibody concentration in the coagulation fluid after the addition of the antibody, and the ability to release phospholipase A2 bound to the cells of each antibody is shown in FIG. 18.
  • Control mouse stakes did not release phospholipase ⁇ 2 from cells at all, but 12 ⁇ 5, 10.1 and 1.4 released phospholipase ⁇ 2 from cells in a concentration-dependent manner.
  • the anti-rest 1.4 H and L chain variable region cDNAs were prepared as shown in Fig. 9 using "5, RACE System Kit” (manufactured by Gibco). That is, the constant region gene (C) and High Priestess head formed to primer first and reverse fc 'enzyme (SUPER SCRIPT II Reverse Transcri ptase) messenger one RNA of 1 ⁇ using ⁇ ;. 4'a manner present; T! CDNA was synthesized.
  • C constant region gene
  • High Priestess head formed to primer first and reverse fc 'enzyme (SUPER SCRIPT II Reverse Transcri ptase) messenger one RNA of 1 ⁇ using ⁇ ;. 4'a manner present; T! CDNA was synthesized.
  • GSP1L (5'-GGCACCTCCAGATGTTAACTGC-3 ') /
  • GSP1H 5'-GGAA (AG) r rA (AGC) CCCTTGACCAGGC -3 ') Z-sequence number 3 ⁇ 4: 12 ".
  • the sequence in parentheses ⁇ reduces the nucleotide corresponding to 3 ⁇ .
  • the mRNA-mRNA was digested with RNaseH, and then ⁇ i'ic DNA was purified using GLASS MAX Spin Cartridgej.
  • a poly (dC) tail was ligated to the T-terminus H chain and Li TJ variable region construction-(V) is an anchor-primer (5, -CUACUACUACUAGGCCACGCGTCGACTAGTACGGGGG) that hybridizes with the poly (dC) tail.
  • the H chain and Ui'inJ variable region cDNA of antibody 10.1 were prepared by the method shown in Fig. 12 using "Marathon cDNA Amplification Kit” (manufactured by Clontechne I :). That is, "M Single-stranded cDNA was synthesized using 1 g of messenger RNA using arathon cDNA synthesis primer and reverse fe enzyme. The reaction solution was further reacted with RNaseH, DNA polymerase I (DNA polymerase), and DNA ligase to form double-stranded cDNA. Furthermore, double-stranded cDNA was blunt-ended by reacting this reaction solution with T4 DNA polymerase (T4 DNA polymerase erase).
  • T4 DNA polymerase erase T4 DNA polymerase erase
  • the Rathon cDNA adapter was ligated to the single-stranded jcDNA using T4 DNA 1 igase.
  • the H- and L-chain variable region promoters (V) are applied to the adapter primer-1 and the primer -GSP2L (the i-chain of the light chain or GSP2H (place of "H"!) Which form a hybrid with the adapter.
  • V variable region promoters
  • the PCR-amplified product was subjected to 1.2% agarose gel II swimming, the band was detected at about 550 to 570 salt ⁇ pair for both Satoshi and the L chain. did it.
  • the PCR amplified fragment was inserted into the pCRT Mini vector.
  • the pCRTMI I plasmid into which the fragment was inserted was transformed into a competent cell of Escherichia coli JM109 (manufactured by ' ⁇ '). ! Play Bok on the> colonies and pCR 1 M II and Haipuri' Bok 'to form "5, primer UD (5' -ACCGAGCTCGGATCCACTAG-3 ' ) 3 ⁇ 4 column *: 16" and "3, primers one DU (5' - ATGCATGCTCGAGCGGCCGCC-3 ') Z sequence ⁇
  • Figure 10 shows the L chain of antibody 1.4
  • ⁇ 11 shows the H chain of antibody 1.4
  • the i-th CD i column of the L chain of antibody 1.4 is 1%! Column number: 5 to 7, and the predicted CDR sequence of the H chain of antibody 1.4 is reduced to SEQ ID NOs: 8 to 10.
  • the antibody 1.4 can be used, for example, for Pag-1 pile rest (Hughes-Jones, NC et.al. Biochem. J., 268, 135-140 (1990)) and WEA anti-rest (Goni, F. et. Proc. Natl. Acad. Sci. USA, 80, 4837-4841 (1983)).
  • Messenger RNA was dry to the cDNA cloning by Shikawa the "QuickPreP Micro mRNA Puri fi cat ion Kit (Pharmacia Biotech Inc.) j, was prepared from hybrid doughs Ma cells lxl0 7 I. That is, cells extraction buffer ( The mixture was turbidly dissolved in an extraction buffer, and oligo dT cellulose (Oligo (dT) -Cellulose) was applied to release messenger RNA.
  • the H-chain and L-promoting variable region cDNA of antibody 12H5 was prepared using the 5 ′ RACE System Kit (GIBC0 BRL) j as shown in FIG. 9; that is, the normal region (C)
  • the iDNA was synthesized by ffl using 1 mg of messenger RNA by ffl using a primer which forms a hybrid and SUPER SCRIPT II Reverse Transcriptase.
  • GSP1L 5 '-GGCACCTCCAGATGTTAACTGC-3'
  • 3 ⁇ 4 In the H chain, it was "GSP1H (5'-GGAA (AG) TA (AGC) CCCTTGACCAGGC-3 ') / sequence number: 12".
  • the sequence in parentheses indicates the base corresponding to the degeneration.
  • the messenger RNA in the form of a gun was digested with RNaseH, and the -strand cDNA was purified using GLASS MAX Spin Cartridge j. Single-strand cDNA was obtained using dCTP and terminal deoxynucleotide transferase.
  • a poly (dC) tail was attached to the 3 'end of the cDNA, and the H and L chain variable region genes (V) were anchor primers that hybridized with the poly (dC) tail (5'-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGI I GGGI IGGGI IG -3, / ffi column «Re: 13) and ⁇ Regional region-Primer that forms a hybrid with (C) GSP2L (5'-TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC-3,) / ft!
  • the reaction products were analyzed by 1% agarose gel electrophoresis, and clones in which amplification of DNA fragments of about 600 to 620 salt pairs were observed were selected.
  • the cloned E. coli was cultivated, and plasmid DNA was prepared using rQIAGEN Plasmid Mini Kit (QIAGEN) j.
  • the prepared plasmid DNA of each clone was purified using the DNA Sequencing Kit (Perkin Elmer) and the 377 DNA Sequencer (Applied Biosystems Inc.) using the primers UD and DU. The nucleobase sequence analysis was performed.
  • Keizo Ushikawa purchased a male BALB / c mouse (4 weeks old, body; 15-20g) from SLC, raised it for 4 yen, and went to the bell.
  • Cisplatin-inducible 1 ⁇ non ⁇ A model was prepared as follows. Specifically, 0.6 ml of cisplatin (Landa, ⁇ ”
  • Rat phospholipase-2 monoclonal pile rest (1.0 mg / 0.2ni1 / mouse) was injected intraperitoneally in the same manner as the anti-human phospholipase-2 monoclonal antibody injection method. Inject 0.6 ml of U'l! & Saline in the back subcutaneously instead of cis-bratin, and inject saline (0.2 ml) with anti-human I and II phospholipase A2 The mice were intraperitoneally administered in the same manner as described in (1) and (2) mice were used.
  • mice After administration of cisplatin, all mice were treated with ether hemp at 3 f: IM, and heparinized 1! Via an abdominal human vein. After that, the abdominal human artery was cut off, the mouse was killed, and the left 3 ⁇ 4 was removed. The blood sample was subjected to 5,000 rpm for 20 minutes, and the BUN was measured using plasma as BUN Kainos (available from Kainos). For 3 ⁇ 4, after removing the capsule, the capsule was removed, and the slice was cut along the red rim.
  • BUN Kainos available from Kainos
  • the slices were incubated for 8 hours with 10% formalin buffer, and then washed with water to remove formalin, and ethanol (70, 80, 90, 99.5, 100%), dehydrate the benzene series, complete the benzene series, and paraffin-saturated benzene.
  • I went to the river.
  • a microtome was cut into a 2 ⁇ m thick tissue section, stained with hematoxylin and eosin (HE), and subjected to string-like examination.
  • the body K of the mice was measured every day during the sudden examination period. First, the body ffi of a normal mouse was about 20 g at the beginning of the test, and about 21 g at the end of the abrupt test, showing an increase in lgB.
  • Blood BUN was reduced to 17.5 mg / d 1 in ⁇ mice. Was increased to 33.9 mg / d 1 to about 2 ⁇ ⁇ by cisplatin administration .
  • Mouse anti-rat type II phospholipase ⁇ 2 monounal suspension was almost similar to cis-platin administration in Ifn serum BUN.
  • the average value of liuil BUN which was obtained by throwing a pile of human phospholipase A2 monoclonal (0.3 mg / mouse), was 27.5 mg / d1, and ⁇ ⁇ About 40% of BUNft that had been exposed to I ⁇ W was suppressed by cis-bratin injection in mice.Pile human phospholipase ⁇ ⁇ ⁇ ⁇ 2 Monochrome-Null stake (l.Omg / mouse)
  • the average plasma BUN in the group was 24.7 mg / dl, which suppressed about 60% of BUNiii'i: increased in rats by cisplatin administration in mice.
  • the cisplatin group and mouse anti-rat type II phospholipase A2 In the case of monochromatic stagnation ⁇ !
  • mice Male BALB / c mice (4 weeks old, body SU5-20g) purchased from SLC, raised one in four, and then submitted to the experiment.
  • Cisplatin induction Injury model mouse was prepared by subcutaneous injection of cisplatin (lander injection, H-Hon Kayaku Co., Ltd., 0.5 ig / ml) 0.6 mU iW subcutaneously to the ⁇ part of the mouse.
  • Control monoclonal anti-reactivity that does not react with mouse 11-phospholipase A2 as a control group (5H8; Sugita Y. et al., I ⁇ imology, 82, p34-41 (1994)) (1.0 mg / 0.2 ml / mouse) ) was injected into the hip cavity in the same manner as in the administration of anti-human type 11 phospholipase A2 monoclonal pile.
  • the urine was centrifuged using i-Rose G rTPTP (manufactured by Diatron) to produce urinary glu-mil-trans-peptidase (7-glutamylyl). transpeptidase) activity was measured and, at the same time, corrected for urine width creatinine content measured using CRE-EN Kainos (manufactured by Kainos Corporation).
  • Phospholipase A2 activity is expressed as a percentage of the amount of tritium oleoleic acid released by hydrolysis of the group added to the reaction mixture (a large intestine 1 cleat product incorporating tritium-labeled oleic acid).
  • the control antibody did not suppress the increase in phospholipase A2 activity.
  • the anti-human type II phospholipase A2 monoclonal antibody administered once 24 hours before cisplatin administration maintained its effect even in the kidney 4 days after cisplatin administration (C1 on 5 days after antibody administration).
  • FIG. 16 shows urinary y-GTP activity on day 1 after administration of cisplatin.
  • A-GTP an indicator of tubular injury
  • Anti-human II 3 ⁇ 4 phospholipase A2 monoclonal antibody injection-/ ⁇ In the case of 0.3H, 12mg administration of 12H5, 1.4mg of 0.1mg, 0.3mg, 1.Omg injection of H-GTP by cisplatin injection The upper bound of activity was significantly reduced by 76%, 87%, 82%, 62% and 84%, respectively. However, the control monoclonal pile did not suppress the upper W of ⁇ -GTP.
  • FIG. 17 shows the plasma BUN i at 4 H after administration of cisplatin.
  • the plasma BUN which is the fingerling of the ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ + ⁇ ⁇
  • the plasma BUN which is the fingerling of the ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ + ⁇ ⁇
  • 0.1%, 0.3mg, l.Orag administration of 12H5; 0.3mg, 1.4Omg injection of 1.4H, and plasma BUN by cisplatin administration in 1.4H injection The increase in ars
  • anti-human type II phospholipase A2 monoclonal antibody can be used as a drug to suppress the onset and progression of cisplatin and dystrophy by inhibiting phospholipase A2.
  • an anti-human ⁇ type phospholipase A2 monoclonal antibody, or a platinum compound of a protein or peptide having a U-harmful potential containing a part thereof was revealed that it can be used as a drug to suppress the onset and progress of renal injury caused by cisplatin administration. That is, it was possible to suppress nephrotoxicity, which is an obvious side effect, of cisplatin. By increasing the dose of cisplatin, it became possible to increase the efficacy of cisplatin.
  • the pharmaceutical composition of the present invention is ffl as an inhibitor of harm caused by administration of a gold compound.
  • Iwakawa is also a prophylactic agent that stops the team by f-dosing before or during administration of gold compounds.
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Tyr lie Arg Tyr Ser Gly Tyr Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 SEQ ID NO: 10
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • N represents inosine (I).
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid

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Abstract

Les problèmes rénaux provoqués par l'administration de cisplatine sont atténués en administrant un anticorps ayant un effet inhibiteur sur la phospholipase A2 du type II. Un anticorps monoclonal capable d'inhiber l'activité de la phospholipase A2 du type II ou des protéines ou des peptides contenant une partie de cet anticorps et ayant le même effet inhibiteur peuvent atténuer la nephrotoxicité qui est l'effet scondaire le plus important de la cisplatine, et qui limite la dose de cisplatine et, par conséquent, peuvent être utilisés comme médicament pour résoudre les problèmes rénaux provoqués par l'administration de cisplatine.
PCT/JP1997/002241 1996-06-27 1997-06-27 Medicament permettant d'attenuer les problemes renaux WO1997049427A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005075647A1 (fr) * 2004-02-06 2005-08-18 Nymox Corporation Anticorps monoclonal anti af-20 humanise
EP1999278A4 (fr) * 2006-03-03 2009-12-09 Univ Southern California Marqueurs génétiques permettant de prédire une affection et l'issue d'un traitement
US20130302326A1 (en) * 2009-04-27 2013-11-14 Case Western Reserve University Pyro-glutamate abeta targeting agents

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996020959A1 (fr) * 1994-12-29 1996-07-11 Yamanouchi Pharmaceutical Co., Ltd. Nouvel anticorps monoclonal ayant un effet inhibiteur sur la phospholipase a2 de type ii, et proteine contenant une partie de cet anticorps

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996020959A1 (fr) * 1994-12-29 1996-07-11 Yamanouchi Pharmaceutical Co., Ltd. Nouvel anticorps monoclonal ayant un effet inhibiteur sur la phospholipase a2 de type ii, et proteine contenant une partie de cet anticorps

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
EUR. J. CLIN. PHARMACOL., 1993, Vol. 44, Suppl. 1, PFEILSCHIFTER J. et al., "Cytokine Regulation of Group II Phospholipase A2 Expression in Glomerular Mesangial Cells", p. S7-S9. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005075647A1 (fr) * 2004-02-06 2005-08-18 Nymox Corporation Anticorps monoclonal anti af-20 humanise
EA010687B1 (ru) * 2004-02-06 2008-10-30 Нимокс Корпорейшн Гуманизированное антитело
EP1999278A4 (fr) * 2006-03-03 2009-12-09 Univ Southern California Marqueurs génétiques permettant de prédire une affection et l'issue d'un traitement
US20130302326A1 (en) * 2009-04-27 2013-11-14 Case Western Reserve University Pyro-glutamate abeta targeting agents
US9109021B2 (en) * 2009-04-27 2015-08-18 Case Western Reserve University Pyro-glutamate Aβ targeting agents

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