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WO2008130204A1 - Nouvel anticorps monoclonal anti-irc85, composition comprenant celui-ci utilisée dans la prévention et le traitement de la tuberculose ou d'une entérite et utilisation - Google Patents

Nouvel anticorps monoclonal anti-irc85, composition comprenant celui-ci utilisée dans la prévention et le traitement de la tuberculose ou d'une entérite et utilisation Download PDF

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Publication number
WO2008130204A1
WO2008130204A1 PCT/KR2008/002334 KR2008002334W WO2008130204A1 WO 2008130204 A1 WO2008130204 A1 WO 2008130204A1 KR 2008002334 W KR2008002334 W KR 2008002334W WO 2008130204 A1 WO2008130204 A1 WO 2008130204A1
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Prior art keywords
irc85
tuberculosis
monoclonal antibody
antibody
new anti
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Byoung Se Kwon
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EUCELL BIO Co Ltd
Immunomics Co Ltd
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EUCELL BIO Co Ltd
Immunomics Co Ltd
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Priority to US12/596,747 priority Critical patent/US8445643B2/en
Priority claimed from KR1020080037968A external-priority patent/KR100900987B1/ko
Publication of WO2008130204A1 publication Critical patent/WO2008130204A1/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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to a new anti ⁇ IRC85 monoclonal antibody; the composition comprising the same for preventing and treating tuberculosis or enteritis disease; and the use thereof.
  • IRC85 (Z39Ig/CRIg, co-receptor of VSIG4, V-set, Ig-domain-containing 4) consists a signal peptide, an extracellular domain having Ig domain, transmembrane domain, and intracellular domain (Langnasese K., Cloning of Z39Ig, a novel gene with immunoglobulin-like domains located on human chromosome X. Biochim Biophys Acta., pp.1492-5225, 2000).
  • IRC85 genes were expressed together with typical complement family in macrophages derived from monocytes as well as mainly the fetal tissues, adult lungs and placentas from human (Walker MG., Z39Ig is co-expressed with activated macrophage genes. Biochim Biophys Acta., pp.1574-3879, 2002).
  • IRC85 is concerned in removing pathogens opsonized with C3 by phagocytosis, or in preventing the infection into another organ through binding with C3b or IC3b, a by-product of the complement C3 as the receptor of the complement C3 which is highly expressed on Kuffer cells in liver (Helmy K.Y., CRIg; a macrophage complement receptor required for phagocytosis of circulating pathogens, Cell, 124(5): pp.915-27, 2006).
  • the anti-IRC85 monoclonal antibody specifically binding to IRC85 could become the main target in developing the therapeutic agent to treat various bacterial diseases such as tuberculosis, enteritis disease etc caused by Tuberculosis, Tubercle bacillus, Yersinia, listeria, Salmonella, Shigella, Legionella, L. monocytogenes and the like (Melanie Hamon. , Listeria monocytogens: a multifaceted model, Nature Reviews microbiology, pp.423-434, 2006).
  • the present inventors have prepared 6H8 hybridoma cell producing novel anti ⁇ IRC85 monoclonal antibody specifically binding with IRC85 and found that the antibody showed potent effect in removing the infected/phagocytosed bacteria from THP-I, a monocytic cell that expresses human IRC85 and is infected with Listeria monocytogenes or MDR-tubercle bacillus to complete the present invention.
  • the present invention provides a new anti-IRC85 monoclonal antibody comprising the sequence with amino acid sequence having SEQ ID No. 5 or more than 90% homology to the amino acid sequence! and the sequence with amino acid sequence having SEQ ID No. 6 or more than 90% homology to the amino acid sequence.
  • the present invention provides a new anti-IRC85 monoclonal antibody comprising the sequence with the nucleotide sequence having SEQ ID No. 7 or more than 90% homology to the nucleotide sequence! and the sequence with nucleotide sequence having SEQ ID No. 8 or more than 90% homology to the nucleotide sequence.
  • the present invention provides a novel Escherichia coli S0LR/6H8-H cell line (Name of depositary institution: Korean Collection for type Cultures, Date of deposit: March 22, 2007, Accession Number: KCTC11097BP) producing the heavy chain of the above-described new anti-ICR85 antibody.
  • the present invention provides a novel Escherichia coli S0LR/6H8- L cell line (Name of depositary institution: Korean Collection for type Cultures, Date of deposit: March 22, 2007, Accession Number: KCTC11098BP) producing the light chain of the above-described new anti-ICR85 antibody.
  • the present invention provides a composition comprising a new anti- IRC85 monoclonal antibody as an active ingredient for preventing and treating tuberculosis disease, together with a pharmaceutically acceptable carrier.
  • the present invention provides a use of new anti-IRC85 monoclonal antibody for the preparation of therapeutic agent for preventing and treating tuberculosis disease in a mammal including human in the need thereof.
  • the present invention provides an immunotherapeutic method for preventing and treating tuberculosis diseases in a mammal comprising administering to said mammal an effective amount of the above-mentioned new anti-IRC85 monoclonal antibody as an active ingredient, together with a pharmaceutically acceptable carrier thereof.
  • the present invention provides a composition comprising a new anti- IRC85 monoclonal antibody as an active ingredient for preventing and treating enteritis diseases, together with a pharmaceutically acceptable carrier.
  • the present invention provides a use of new anti-IRC85 monoclonal antibody for the preparation of therapeutic agent for preventing and treating enteritis disease in a mammal including human in the need thereof.
  • the present invention provides immunotherapeutic method for preventing and treating enteritis disease in a mammal comprising administering to said mammal an effective amount of the above-mentioned new anti-IRC85 monoclonal antibody as an active ingredient, together with a pharmaceutically acceptable carrier thereof.
  • the present invention provides a health care food comprising a new anti-IRC85 monoclonal antibody as an active ingredient for preventing and improving tuberculosis disease.
  • the present invention provides a health care food comprising a new anti-IRC85 monoclonal antibody as an active ingredient for preventing and improving enteritis disease.
  • ⁇ 22> Accordingly, it is an object of the present invention to provide a new anti ⁇ IRC85 monoclonal antibody comprising the sequence with amino acid sequence having SEQ ID No. 5 or more 90% homology in the amino acid sequence; and the sequence with amino acid sequence having SEQ ID No. 6 or more than 90% homology to the amino acid sequence.
  • the present invention provides a new anti-IRC85 monoclonal antibody consisting of amino acid sequences having SEQ ID No. 5 or SEQ ID No. 6.
  • a new anti-IRC85 monoclonal antibody comprising the sequence with the nucleotide sequence having SEQ ID No. 7 or more than 90% homology to the nucleotide sequence; and the sequence with nucleotide sequence having SEQ ID No. 8 or more than 90% homology to the nucleotide sequence.
  • the present invention provides a new anti-IRC85 monoclonal antibody coded by the sequences consisting of the nucleotide sequences having SEQ ID No. 7 and SEQ ID No. 8.
  • the term "new anti-IRC85 monoclonal antibody” disclosed herein may comprise the heavy chain comprising at least one polypeptide selected from the group consisting of TSGMGVG (HCDRl), DIFWDDNKYYNPSLKS (HCDR2), VYYKNDGYFD (HCDR3), VTVPSSTW and SVSELPIM, preferably, TSGMGVG (HCDRl), DIFWDDNKYYNPSLKS (HCDR2) and VYYKNDGYFD (HCDR3).
  • the term "new anti-IRC85 monoclonal antibody” disclosed herein may comprise the light chain comprising at least one polypeptide selected from the group consisting essentially of RASKSVTTSGYSFMH (LCDRl), LASNLEP (LCDR2), QHSRELPYT (LCDR3), FYPKDIN and STYSMS, preferably, RASKSVTTSGYSFMH (LCDRl), LASNLEP (LCDR2) and QHSRELPYT (LCDR3).
  • the term "new anti-IRC85 monoclonal antibody” disclosed herein shall be understood in the art that the amino acid sequence of the heavy chain includes the other heavy chain variants substituted with the part or sole of the heavy chain amino acid sequence specifically binding to the IRC85 derived from a human, dog, cow, or pig, preferably, human.
  • the term "new anti-IRC85 monoclonal antibody” disclosed herein shall be understood in the art that the amino acid sequence of the light chain includes the other light chain variants substituted with the part or sole of the heavy chain amino acid sequence specifically binding to the IRC85 derived from a human, dog, cow, or pig, preferably, human.
  • the present invention also provides a novel Escherichia coli S0LR/6H8-H cell (Name of depositary institution: Korean Collection for type Cultures, Date of deposit: March 22, 2007, Accession Number: KCTC11097BP) producing the heavy chain of the above-described new anti-ICR85 antibody.
  • the present invention provides a new Escherichia coli S0LR/6H8-L cell (Name of depositary institution: Korean Collection for type Cultures, Date of deposit: March 22, 2007, Accession Number: KCTC11098BP) producing the light chain of the above-described new anti-ICR85 antibody.
  • a novel Escherichia coli S0LR/6H8-H cell comprises the cells prepared the method comprising the steps of; preparing CDNA library by isolating mRNA from 6H8-hybridoma cell producing novel anti-
  • IRC85 monoclonal antibody at 1 step searching and detecting the cDNA expressing the light chain of 6H8 antibody by using the constant region of antibody, preferably, CHl domain of mouse IgGl cDNA from the cDNA library of the 1 step as a probe at the 2 step; and transforming the detected 6H8-H
  • a novel Escherichia coli S0LR/6H8-L cell disclosed herein comprises the cells prepared the method comprising the steps of; preparing CDNA library by isolating mRNA from 6H8-hybridoma cell producing novel anti-
  • IRC85 monoclonal antibody at 1 step searching and detecting the cDNA expressing the light chain of 6H8 antibody by using the constant region of antibody, preferably, CHl domain of mouse kappa-chain cDNA from the cDNA st nd library of the 1 step as a probe at the 2 step; and transforming the nd detected 6H8-L prepared in the 2 step into a plasmid through in vivo rd excision using by Exassit SOLR system to identify the sequence at the 3 step.
  • the present invention provides a pharmaceutical composition comprising a new anti-IRC85 monoclonal antibody as an active ingredient for preventing and treating tuberculosis disease, together with a pharmaceutically acceptable carrier.
  • the present invention provides a use of new anti-IRC85 monoclonal antibody for the preparation of therapeutic agent for preventing and treating tuberculosis disease caused by in a mammal including human in need thereof.
  • the present invention provides an immunotherapeutic method for preventing and treating tuberculosis diseases in a mammal comprising administering to said mammal an effective amount of the above-mentioned new anti-IRC85 monoclonal antibody as an active ingredient, together with a pharmaceutically acceptable carrier thereof.
  • the present invention provides a composition comprising a new anti- IRC85 monoclonal antibody as an active ingredient for preventing and treating enteritis disease, together with a pharmaceutically acceptable carrier.
  • the present invention provides a use of new anti-IRC85 monoclonal antibody for the preparation of therapeutic agent for preventing and treating enteritis disease in a mammal including human in need thereof.
  • the present invention provides an immunotherapeutic method for preventing and treating enteritis diseases in a mammal comprising administering to said mammal an effective amount of the above-mentioned new anti-IRC85 monoclonal antibody as an active ingredient, together with a pharmaceutically acceptable carrier thereof.
  • the present invention provides a health care food comprising a new anti-IRC85 monoclonal antibody as an active ingredient for preventing and improving tuberculosis disease.
  • the present invention provides a health care food comprising a new anti-IRC85 monoclonal antibody as an active ingredient for preventing and improving enteritis diseases.
  • the term “tuberculosis disease” disclosed herein comprises MDR- tuberculosis, lung tuberculosis, cystica tuberculosis, bone tuberculosis, guttural tuberculosis, lymph tuberculosis, breast tuberculosis or spinal tuberculosis, preferably, MDR-tuberculosis, which is caused by tuberculosis virulent germ.
  • enteritis virulent germ a malignant neoplasm originating from enteritis virulent germ.
  • enteritis virulent germ preferably, Crohn's disease, which is caused by enteritis virulent germ.
  • the term "tuberculosis virulent germ” disclosed herein comprises the germ selected from Mycobacterium tuberculosis, tubercle bacillus, or
  • infectious germ comprises the germ belonged to the genus selected from Yersina, Listeria, Salmonella, or Shigella, specifically, Listeria or Salmonella.
  • inventive anti-IRC85 monoclonal antibody of the present invention may be prepared by the method well-known in the art (Ed Harlow, Antibodies; A Laboratory manual, Cold Sproing Harbor laboratory press, ppl96-218), and exemplary method is shown as follows:
  • Present invention provides a method for preparing the inventive anti-IRC85 monoclonal antibody by the method comprising the steps consisting of: injecting IRC85- expressing cell line (1-1C2) BALB/c mouse four times for every other weeks to
  • St immunization at the 1 step isolating B lymphocyte from the spleen of the nd immunized mouse at the 2 step; mixing the B lymphocyte with myeloma cell such as Sp2/0-Agl4 with the mixed ratio of 3:1 to fuse in DMSO medium comprising 50% PEG for 1 minute and washing with DMEM medium supplemented with high-concentrated sugar which was diluted with 50% PEG at the 3 step; suspending the fused cells in HAT medium to select the hybridoma cells fused with only B lymphocyte and S ⁇ 2/0-Agl4 at the 4 step; and selecting 6H8 hybridoma cell producing new anti-ICR85 monoclonal antibody from the hybridoma cells.
  • myeloma cell such as Sp2/0-Agl4
  • the present invention provides a composition comprising a new anti-IRC85 monoclonal antibody prepared by the above-described method as an active ingredient for preventing and treating enteritis disease, together with a pharmaceutically acceptable carrier.
  • the present invention provides a use of new anti-IRC85 monoclonal antibody prepared by the above-described method for the preparation of therapeutic agent for preventing and treating enteritis disease in a mammal including human in need thereof.
  • the present invention provides an immunotherapeut ic method for preventing and treating enteritis diseases in a mammal comprising administering to said mammal an effective amount of the above-mentioned new anti-IRC85 monoclonal antibody prepared by the above-described method as an active ingredient, together with a pharmaceutically acceptable carrier thereof .
  • the inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, Easton PA).
  • the inventive composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrol idone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.
  • pharmaceutically acceptable carriers, adjuvants or diluents e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium
  • the formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like.
  • the compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.
  • compositions of the present invention can be dissolved in oils, propylene glycol or other solvents which are commonly used to produce an injection.
  • suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them.
  • the compounds of the present invention can be formulated in the form of ointments and creams.
  • compositions containing inventive composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), suppository, or sterile injectable preparation (solution, suspension, emulsion).
  • inventive composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
  • the desirable dose of the inventive composition varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging 0.0001 to 100 mg/kg, preferably, 0.001 to 10 mg/kg by weight/day of the inventive composition of the present invention.
  • the dose may be administered in a single or multiple doses per day.
  • inventive composition can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectal Iy or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intracerebroventricular injection.
  • a health care food comprising the above described antibody preventing and improving tuberculosis disease or enteritis diseases.
  • the amount of above described composition in food or beverage may generally range from about of total weight of food for the health beverage composition and 0.02 to 10 g, preferably 0.3 to 1 g on the ratio of 100 ml of the health beverage composition.
  • the health beverage composition of present invention contains above described composition as an essential component in the indicated ratio
  • the other component can be various deodorant or natural carbohydrate etc such as conventional beverage.
  • natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc! conventional sugar such as dextrin, cyclodextrin! and sugar alcohol such as xylitol, and erythritol etc.
  • natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al .
  • the amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 ml of present beverage composition.
  • the other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al.
  • the other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination.
  • the ratio of the components is not so important but is generally range from about 0 to 20 w/w% per 100 w/w% present composition.
  • Examples of addable food comprising aforementioned composition therein are various food, beverage, gum, vitamin complex, health improving food and the like.
  • Inventive anti ⁇ IRC85 monoclonal antibody of present invention showed potent anti-bacterial activity against Listeria mnocytogenes and Tubercle bacillus, therefore it can be useful as a composition for treating and preventing tuberculosis disease and enteritis disease.
  • Fig. 1 represents the vector-map in IRC85/pcDNA3.1
  • Fig. 2 represents the expression of IRC85 cell
  • Fig. 3 presents the expression and purification of IRC85 protein from
  • Fig.4 presents the FACS analysis of inventive monoclonal antibody
  • Fig. 5 depicts the expression of IRC85 in THP-I cell
  • Fig. 6 presents the removal rate of anti-human IRC85 monoclonal antibody against Listeria monocytogenes according to various concentrations
  • Fig. 7 depicts the removal rate of anti-human IRC85 monoclonal antibody against MDR (multi-drug resistance) tuberculosis according to various concentrations.
  • MDR multi-drug resistance
  • Example 1 Preparation of HeLa cell expressing IRC85 ⁇ 92> 1-1. IRC85 cloning using pCDNA3.1 ⁇ 93> To express IRC85 in animal cells, pCDNA3.1 (Invitrogen Co. Ltd., See Fig. 1) was used and full-length of IRC85 was prepared to use by PCR for cloning vectors. The primers of IRC85 was shown in following Table 1.
  • IRC85 sense SEQ ID No. 1 5'-CGGGATCCGAA ⁇ CGGTACCCGTCCCATCCTG
  • IRC85 anti-sense SEQ ID 5'-CGGGATCCGAATTCGGTACGATGAAGTTGCCA ⁇ 98> No. 2 TTCTGCCTGCC-3'
  • PCR reaction was performed as follows: 1 cycle for 5 mins at 94 0 C; 8 cycles for 30sec at 94 0 C, for 30sec at 50°C, for 45 sec at 72°C; 30 cycles for 30 sec at 94°C , 30 sec for 65°C , 45 sec at 72°C ; 1 cycle for 5 mins at 72 °C.
  • pcDNA 3.1 and PCR byproduct were excised by restriction enzyme BamH I/Not I, and the excised DNA was confirmed by electrophoresis developed with 1% agarose gel to extract using by Gel extraction kit (QIAGEN, QIAquick Gel extraction kit, Cat# 28706).
  • the extracted vector and PCR product was mixed with the ratio of 1:3, and 10 ⁇ i of 2X ligation buffer, and 1 ⁇ i of T4 DNA ligase (100 U/ml) were added thereto to adjust the final concentration 20 ⁇ i.
  • the synthetic reaction was performed at room temperature for 2 hours, and ligate was mixed with 200 ⁇ l of DH5 ⁇ competent cell thoroughly to be left alone in iced water for 45 mins and then heated at 42 0 C for 90sec.
  • the reaction solution was left alone in iced water for 5 mins and 500 ⁇ i of IX LB medium was added thereto to incubate at 37 ° C for 60 mins. After the incubation, the solution was centrifuged with the speed of 14000 rpm for 10 sec and 100 ⁇ i of supernatant was collected.
  • the cell pellet was suspended in the supernatant and the cell was cultured for 16 hours by spreading onto IX LB/Amp (100 ⁇ g/ml) plate to transformation.
  • the colony grown in Ampicillin selection broth was inoculated into LB/Amp broth to incubate and isolate plasmid DNA.
  • Plasmid DNA was extracted from the inoculated bacteria in Example 1-1 according to Alkaline lysis method. The bacteria were collected and suspended in 100 ⁇ Jt of mixture solution I (50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8.0). 200 id of solution ⁇ (0.2 N NaCl, 1% SDS) was added to give rise to soft inverting and left alone for 5 mins at room temperature. 5 mins after the incubation, solution JR (3 M Potassium acetate, glacial acetic acid) was added thereto to be left alone in ice for 5 mins, and centrifuged with the speed of 14000 rpm for 10 mins.
  • mixture solution I 50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8.0
  • 200 id of solution ⁇ 0.2 N NaCl, 1% SDS
  • solution JR 3 M Potassium acetate, glacial acetic acid
  • the supernatant was transferred to new tube, and two fold volume of ethanol was added thereto to centrifuge again with the speed of 14000 rpm for 10 mins. After the centrifugation, the supernatant was discarded, washed with ImI of 70% ethanol, and the plasmid DNA was dried in the air to dissolve in deionized solution. 3 ⁇ i of the prepared DNA was excised by restriction enzyme BamH I/Not I for 2 hours at 37°C to perform electrophoresis on 1% agarose gel for 2 hours and confirm the insertion to cloning.
  • Example 2 Transfect ion of IRC85/ ⁇ cDNA3. 1 into HeIa cel l ⁇ iO7> 2-1. Culture of HeIa cel l ⁇ iO8> HeIa cell (ATCC) was cultured in high glucose DMEM (Dulbeccos Modified Eagle Medium, GIBCO/BRL) supplemented with 10% FBS (fetal bovine serum), penicillin (100 IU/ml) and streptomycin (100 ⁇ i/m ⁇ ) at 37°C in 5% CO 2 incubator.
  • DMEM Dulbeccos Modified Eagle Medium, GIBCO/BRL
  • FBS fetal bovine serum
  • penicillin 100 IU/ml
  • streptomycin 100 ⁇ i/m ⁇
  • ⁇ ii2> One day before the transfection, 5x10 cells were plated onto 6-well plate and the cells were replaced with new broth one hour before transfection. For transfection, 10 ⁇ g of DNA was added to 84.5 ⁇ i of DDW and mixed with 12.5 ⁇ of 2.5 M calcium chloride. Identical volume of 2X HBS (280 mM sodium chloride, 10 mM potassium chloride, 1.5 mM disodium hydrogen phosphate, 2 mM dextrose, 50 mM HEPES, pH 7.05) was added thereto dropwisely with vortexing and the mixture was left alone for 20 mins at room temperature to transfer into each well. 6 hours after transfection, cells were washed with PBS and the medium was replaced with new medium.
  • 2X HBS 280 mM sodium chloride, 10 mM potassium chloride, 1.5 mM disodium hydrogen phosphate, 2 mM dextrose, 50 mM HEPES, pH 7.05
  • IRC85-His recombinant protein ⁇ 125> pET21-(a)+/85ext prepared in Example 3-1 was transformed into BL21 (DE3) E. coli and the protein was expressed using 0.1 mM IPTG.
  • the expressed recombinant protein was isolated by using by Ni-NTA resin since it comprises 6X His tag. It expressed 26 KDa by comprising 25 kDa polypeptide expressed from the 700 bp DNA located in IRC85 extracellular domain and 66 Da His tag.
  • Fig. 3 showed that the practically isolated IRC85 using Ni-NTA resin, has 27 KDa with low level of expression since IRC85 extracellular domain mainly consists of hydrophobic amino acids (See Fig. 3).
  • the isolated protein was used as an antigen for ELISA screening during the preparation of monoclonal antibody.
  • Example 4 Preparation of the monoclonal antibody against anti ⁇ IRC85 ⁇ 128> 4-1.
  • Mouse immunization ⁇ 129> 1-1C2 cell line, a cell line expressing IRC85 (IRC: Immunomodulation Research Center) was injected into 4-weeks BALB/c mouse for 2 weeks in a dose of 2X10 cells/mouse four times. 3 days after the 4 injection, the mouse tail was cut to collect blood and the titer of antibody was determined.
  • ELISA was performed using by IRC85-His protein and the dilution less than 1/100000 of the serum showed significance. Fusion was performed by using the mouse.
  • ⁇ i32> Sp2/0-Agl4 cell line was incubated in high glucose DMEM (Dulbeccos Modified Eagle Medium, GIBCO/BRL) supplemented with 10% fetal bovine serum, penicillin (100 IU/ml) containing streptomycin (100 f d/ml) at 37°C, in 5% CO 2 incubator.
  • DMEM Dulbeccos Modified Eagle Medium, GIBCO/BRL
  • penicillin 100 IU/ml
  • streptomycin 100 f d/ml
  • the cells were transferred to 50 ml of conical tube and the tube was full with serum-free DMEM medium to centrifuge for 5 mins with the speed of 1000 rpm. After twice washing, the supernatant was discarded and RBC was removed according to hypotonic lysis method. The tube was full with medium and centrifuged. During the final washing step, Sp2/0-Agl4 myeloma cells were collected.
  • the spleen lymphocyte and SP2/0 myeloma cells were suspended again in medium, and the number of cells was counted using by hematocytometer .
  • the mixture of the spleen lymphocyte and Sp2/0 myeloma cells (3:1) in 50 ml of conical tube was centrifuged with speed of 800 rpm for 5 mins.
  • 1 ml of 50% PEG (polyethylene-glycol 3000 in DMSO, Sigma Co.) was added thereto with vortexing to perform fusion, and the added PEG solution was washed by following diluting steps with high glucose DMEM: twice for 1 ml/min, twice for 5 ml/min, and twice for 10 ml/min. After further washing steps for two times, the fused cells were suspended in HAT-containing medium and distributed to 96-well plates for selection of fused cells. ⁇ 136>
  • ELISA Enzyme-linked immuno sorbent assay Screening ⁇ 138> IRC85-His protein purified from pET21-(a)+/85ext in Example 3-2 was diluted with coating buffer (0.03 M sodium carbonate, 0.068 M sodium bicarbonate, pH 9.4) to the final concentration be 1 ⁇ g/ml , and distributed onto ELISA- 96 well plates by 100 ⁇ fc/well to coat at 4°C for overnight. The supernatant was removed and 3% skim milk was distributed by 100 ⁇ l to incubate at 37 ° C for 1 hour. PBS-T (0.05% Tween-20 in PBS) was distributed onto the plates by 200 ⁇ l and washed three times.
  • the supernatant of fused cells was distributed onto each cell by 100 ⁇ l to react for 2 hours at room temperature. PBS-T was added thereto by 200 ⁇ l and the plates were washed four times. The diluted anti-mouse IgG-AP with PBS-T to 1:5000, was distributed onto the plates by 100 ⁇ /well to incubate at 37°C for one hour. The plates were washed with PBS-T three times and 1 mg/ml of phosphatase substrate (Sigma Co., 104-0) dissolved in carbonate buffer (0.1 M sodium carbonate, 1 mM magnesium chloride, pH 9.8) was distributed onto each well by 50 and incubated at 37°C for 30 mins in the shadow. After the reaction, the absorbance was determined at OD ⁇ nm to select positive clone-
  • anti-IRC85 monoclonal antibody 6H8 (designated as "6H8" hereinafter) expressed as amino acid sequence of SEQ ID No. 5 and 6, or encoded thereof by nucleotide sequences of SEQ ID No. 7 and 8 showed most potent binding activity (See Fig.4).
  • FITC-binding anti-IRC85 monoclonal antibody was prepared according to the standard manual using FITC (Cat# 46110, Pierce). Human Ab antibody was added to the collected cells to block the Fc receptor and the cell was stained with FITC-binding anti-human IRC85 monoclonal antibody for 30 mins to perform flow cytometry analysis (Hohn E., Short Protocols in Immunology, pp.5-23, 2005).
  • THP-I cell (ATCC, 4xlO 5 cells/ml) was induced to be infected with 10 MOI of L. monocytogenes (ATCC 19111, KTCC) at 37°C for 1 hour. After the phagocytosis, the cell was centrifuged for 10 mins with speed of 600xg, washed with PBS and the steps were repeated four times to remove only the cells which are not phagocytized. 300 ⁇ g/ml of ampicillin was added thereto to incubate at 37°C for 15 mins and the adhered bacteria was removed. The infected cell was distributed to 24-well plates and treated with anti-IRC85 or mouse IgG antibody to collect the cells at every hour.
  • the cell was once washed with PBS, dissolved in distilled water and performed to plating on BHIB (brain heart infusion broth; Difco Laboratories, Detroit, MI) medium according to dilution method. More than 12 hours after the incubation at 37 °C , the values of CFU (colony forming unit) and removal ratio were determined by counting the number of colony observed.
  • BHIB brain heart infusion broth
  • THP-1 cell (ATCC, 4xlO 5 cells/ml) was infected with 10 MOI of MDR tubercle bacillus at 37°C for 1 hour. After the end of phagocytosis, the cell was centrifuged with speed of 600xg for 10 mins to collect the infected cells, washed with PBS and the steps were repeated three times to remove the cell which had not been phagocytosized. The cell was treated with 5 ⁇ g/ml of mlgG or anti ⁇ IRC85 antibody, incubated at 37 ° C, and the cell was collected at every hour to form colony by plate incubating on H9 agar plate. The removal ratio of MDR tubercle bacillus due to the anti ⁇ IRC85 monoclonal antibody was determined.
  • RNA was extracted from the cell line producing 2x10 6H8 according to the instruction manual (Easy-Blue Isolation Kit, Intron Co.). ⁇ 168> The cell pellet was treated with Easy-Blue Isolation Kit reagent (Easy-Blue Isolation Kit, Intron Co.) and extracted with phenol/chloroform once. The supernatant was treated with isopropanol to precipitate RNA, washed with 75% ethanol twice and dissolve in distilled water treated with DEPC (Sigma Co.) to obtain total RNA. The extraction of mRNA from the total RNA was isolated and purified by using Dynalbeads mRNA purification kit (DYNAL Co.).
  • SM buffer solution 100 mM sodium chloride, 10 mM magnesium sulfate, 50 mM Tris-HCl pH 7.5, 0.01% gelatin
  • E CoIi XL-I blue MRF' (Stratagene Co. Ltd) was used as a host cell, and the colony obtained by spreading on LB/tetracycline (50 ⁇ g/ml) plate, was inoculated to LB medium containing 10 mM magnesium sulfate and 0.2% maltose.
  • the cell was bred with stirring at the speed of 200 rpm at 37°C on condition that OU6oo had not exceeded 1.0.
  • the culture medium was centrifuged at the speed of 500xg at 10 mins to remove the medium, and diluted by adding 10 mM magnesium sulfate to the cell pellet to the extent that OD 60 O had reached to
  • the host cell was stored at 4°C to use for 48 hours.
  • K-IU 5'- atcttcccaccatccagtga-3' of SEQ ID No. 12 and K-ID: 5'-cgtccttggtcaacgtgag-3' of SEQ ID No. 13 were used as PCR primer for obtaining a light chain probe to prepare the CHl domain of mouse kappa chain cDNA, 200 bp PCR product was obtained to use as a probe.
  • the hydridization was performed by using ECL direct nucleic acid labelling and detection kit (#RPN3000, Amersham), and the clone prepared thereby was transformed to plasmid to confirm the sequence through in vivo excision using Exassit/SOLR system.
  • the amino acid sequence and nucleotide sequence of the heavy chain gene and kappa chain was shown in following Table 3.
  • AAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAA CCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAC ⁇ CGCC
  • Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2 ml ample and sterilizing by conventional injection preparation method.
  • Powder preparation was prepared by mixing above components and filling sealed package.
  • Anti-IRC85 monoclonal antibody (SEQ ID No. 5 and 6) 10 mg
  • Ant i-IRC85 monoclonal ant ibody (SEQ ID No . 5 and 6) 10 mg
  • ⁇ 2ii> Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.
  • Liquid preparation was prepared by dissolving active component, and then filling all the components in 1000 ml ample and sterilizing by
  • Anti-IRC85 monoclonal antibody (SEQ ID No. 5 and 6) 1000 mg
  • Anti-IRC85 monoclonal antibody (SEQ ID No. 5 and 6) 1000 mg
  • Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85°C for 1 hour, filtered and then filling all the components in 1000 ml ample and sterilizing by conventional health beverage preparation method.
  • the novel anti ⁇ IRC85 monoclonal antibody specifically binding with IRC85 showed potent effect in removing the infected/phagocytosed bacteria from THP-1, a monocytic cell that expresses human IRC85 and is infected with Listeria monocytogenes or WR-tubercle bacillus. Accordingly, it can be useful as a medicament and health care food in the prevention and treatment of tuberculosis disease and enteritis disease.
  • CGGGATCCGAATTCGGTACGATGAAG ⁇ GCCATTCTGCCTGCC-S 1 is antisense primer of ICR85
  • SEQ ID. No. 3 5'-CGCGGATCCCAGCAGGCAAAGTACCAG-S' is sense primer of ICR85-
  • GK is heavy chain amino acid sequence of novel anti ⁇ ICR85 antibody, SEQ ID.
  • AGCACCCAGCACTGCC ⁇ GNGACCCTGCNAANAAAAAAAAAAAAAAAAAAAAACTCGAG is heavy chain amino acid of novel ant i-ICR85 ant ibody, SEQ ID . 8 :
  • AAAGTGAGTCTTTGCACTTGAAAAAAAAAAAAAAAAAAAAACTCGAG is light chain amino acid sequence of novel anti-ICR85 antibody
  • SEQ ID. 9 5'- gagagagagagagagagagaactagtctcgagtt-3' is Xhol cognition nucleotide sequence
  • SEQ ID. 10 5'-gaactctggatccctgtcca-3' is CHl domain binding G-IU primer of mouse IgGl cDNA
  • SEQ ID. 11: 5'-tgcaaggcttacaaccacaa ⁇ 3' is CHl domain binding G-ID primer of mouse IgGl cDNA, SEQ ID.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un nouvel anticorps monoclonal anti-IRC85 lié de manière spécifique à l'IRC85 et présentant un puissant effet destructeur des bactéries infectées/phagocytées de THP-I, une cellule monocytique exprimant l'IRC85 humain et infectée par des monocytogènes de Listeria ou un bacille tuberculeux WR. Il peut ainsi être utilisé comme médicament ou produit alimentaire diététique dans la prévention et le traitement de la tuberculose ou d'une entérite.
PCT/KR2008/002334 2007-04-24 2008-04-24 Nouvel anticorps monoclonal anti-irc85, composition comprenant celui-ci utilisée dans la prévention et le traitement de la tuberculose ou d'une entérite et utilisation Ceased WO2008130204A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/596,747 US8445643B2 (en) 2007-04-24 2008-04-24 Anti-IRC85 monoclonal antibody; and composition comprising the same for preventing and treating tuberculosis or enteritis disease; and the use thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2007-0039791 2007-04-24
KR20070039791 2007-04-24
KR1020080037968A KR100900987B1 (ko) 2007-04-24 2008-04-24 신규 항-ｉｒｃ85 단일클론 항체 및 이를 유효성분으로함유하는 결핵 또는 장염 질환의 예방 및 치료용 조성물
KR10-2008-0037968 2008-04-24

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WO2020069507A1 (fr) * 2018-09-28 2020-04-02 Eutilex Co., Ltd. Anticorps anti-vsig4 humains et leurs utilisations
RU2776638C1 (ru) * 2018-09-28 2022-07-22 Ютайлекс Ко., Лтд. Антитела против vsig4 человека и их применение
JP2023527410A (ja) * 2020-05-29 2023-06-28 ヴェルソー セラピューティクス， インコーポレイテッド 骨髄細胞炎症性表現型を調節するための抗ｖｓｉｇ４組成物及び方法、ならびにその使用

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Title
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KIM J.K. ET AL.: "Characterization of monoclonal antibody specific to the Z39Ig protein, a member of immunoglobulin superfamily", IMMUNOLOGY LETTERS, vol. 99, no. 2, 15 July 2005 (2005-07-15), pages 153 - 151, XP004973586 *
LEE M.Y. ET AL.: "Z39Ig is expressed on macrophages and may mediated inflammatory reactions in arthritis and atherosclerosis", JOURNAL OF LEUKOCYTE BIOLOGY, vol. 80, no. 4, 1 August 2006 (2006-08-01), pages 922 - 928 *
SONG Y.M.: "Identification of conterpart of IRC85Z39Ig/CRIg/VSIG4 and in vitro killing of intracellular Listeria monocytogens by IRC85 signaling on monocytes", MASTER OF SCIENCE THESIS, UNIVERSITY OF ULSAN(REP. OF KOREA), DEPARTMENT OF IMMUNOLOGY AND BIOMEDICINE, February 2007 (2007-02-01) *
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020069507A1 (fr) * 2018-09-28 2020-04-02 Eutilex Co., Ltd. Anticorps anti-vsig4 humains et leurs utilisations
US10927183B2 (en) 2018-09-28 2021-02-23 Eutilex Co., Ltd. Anti-human VSIG4 antibodies and uses thereof
CN113423728A (zh) * 2018-09-28 2021-09-21 优特力克斯有限公司 抗人vsig4抗体及其应用
JP2022500484A (ja) * 2018-09-28 2022-01-04 ユーティレックス カンパニー リミテッド 抗ヒトｖｓｉｇ４抗体およびその使用
RU2776638C1 (ru) * 2018-09-28 2022-07-22 Ютайлекс Ко., Лтд. Антитела против vsig4 человека и их применение
EP3873929A4 (fr) * 2018-09-28 2022-11-23 Eutilex Co., Ltd. Anticorps anti-vsig4 humains et leurs utilisations
US11905334B2 (en) 2018-09-28 2024-02-20 Eutilex Co., Ltd. Anti-human VSIG4 antibodies and uses thereof
CN113423728B (zh) * 2018-09-28 2024-03-01 优特力克斯有限公司 抗人vsig4抗体及其应用
JP7535311B2 (ja) 2018-09-28 2024-08-16 ユーティレックス カンパニー リミテッド 抗ヒトｖｓｉｇ４抗体およびその使用
AU2019351294B2 (en) * 2018-09-28 2025-07-31 Eutilex Co., Ltd. Anti-human VSIG4 antibodies and uses thereof
JP2023527410A (ja) * 2020-05-29 2023-06-28 ヴェルソー セラピューティクス， インコーポレイテッド 骨髄細胞炎症性表現型を調節するための抗ｖｓｉｇ４組成物及び方法、ならびにその使用

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