llllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll · 2013. 5. 27. · PatentApplicationPublication Feb．15，2007 Sheet70f7 US2007／003（i734Al

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（19）United States

（12）PatentApplicationPublication（10）Pub．No．‥US2007／0036734Al l肋haraetal．（43）Pub．Date： Feb．15，2007

（54）TIIERAPEUTICAGENTFORPERIODONTAL DISEASE

（75）Inventors：TomoyukiTahara，Gunma（JP）； YoshimitsuAbiko，Tokyo（JP）； IIiromasaYoshie，Niigata（JP）；Tbtsuo Kobayashi，Niigata（JP）；Tbshio Umemoto，Kanagawa（JP）；Nobushiro IIamada，Kanagawa（JP）

CorrespondenceAddress： FOLEYAND LARDNER LLP

SUITE500

3000K STREET NW ⅥASHINGTON，DC20007（US）

（73）Assignees：Kirin Beer KabushikiKaisha；Nihon University

（21）Appl．No．： 10／549，289

（22）PCTFiled： Mar．17，2004

（86）PCTNo．： PCT／JPO4／03569

さ371（c）（1），（2），（4）Date：Aug．14，2006

（30） Foreign Application Priority Data

Mar．17，2003 （JP）………………………………‥2003－072714

Publication Classification

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424／164．1；435／252．33；435／326； 435／254．2

（57） ABSTRACT

ThepresentinventionprovidesahumanmOnOClonalanti－ bodythathasbothactivityofinhibitingtheaggregationof Pathogenic bacteriainvoIvedin periodontaldiseases and activityofpromoting sterilization byleukocytes，thatis Prefbrably a monoclonalantibody agalnSt40－kDa OMII Whichinhibits the binding ofhemin，andthat causes no COnCernSSuChassidee正己cts．Thepresentinventionfurther provides an agent that acts against periodontal diseases COntainlngthemonoclonalantibody．Thepresentinvention relatestoanantibodybindingto40－kDaOMPorafunc－ tionalfragmentthereofhavingatleastoneof（1）activityof inhibitingthecoaggregationofPgingivalis，（2）activityof PrOmOtinghumanneutrophilicphagocytosis，and（3）activity Ofinhibitingthebindingofheminto40－kDaOMP．

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US2007／0036734Al Feb．15，2007

to e正己ctively prevent or treat periodontaldiseases，1tis

importanttosuppresscolonizationofPginglValissoasto eliminatethebacterialbodiesfromwithinperiodontalpock－ ets．

［0005］AsamethodfbrdestroyingPgingivalis，antibody therapymaybeemployed．SuchantibodytherapylnVOIves PreParlngaSPeCificantibodyagalnStaPathogenicfhctorof Pathogenic bacteriainvoIvedin periodontaldiseases and thenadministerlngtheantibodytoaperiodontalpocket．By SuChantibodytherapy，1）suppressionofpathogenicbacte－ rialcolonizationintheperiodontalpocket，2）promotionof Steri1izationbyleukocytes withinperiodontalpocket，and thelikeareexpected・Regardingl），ithasbeenePOrtedthat althoughanantlgenWaSunidentified，re－COlonlZationofP glnglValiscouldbesuccessfu11ysuppressedwithintheperi－ Odontalpocketfbr9monthsthroughlocaladministrationof anantibody againstP gingivalis（Booth，V et al．，Infbct Immun．，1996Ⅵ）164：422）．Asdescribedabove，Periodontal diseasesmaybeovercometosomeextentthroughsuppres－ Sion of colonization ofpathogenic bacteriainvoIvedin PeriodontaldiseasesuslngSuChspecificantibody．Moreover， WhenitisimpossibletoapplylnVaSivetreatmenttoacase Ofseverelyinfectiousperiodontaldiseases，antibodytherapy Wherebyactivityofpromotingsteri1ization（activityofpro－ motingphagocytosis）byleukocyteswithintheperiodontal POCketisactivelyexpectedmayalsobenecessary．

［0006］Recently，40－kDaoutefmembraneprotein（OMP） hasbeenreportedasoneofantlgenSeXPreSSedinPgingl－ Valis（Abiko，Yet．al．，Arch．Oral．Biol．1990Ⅵ）135：689， KawamOtO，Yetal．，Int．J．Biochem．，1991Ⅵ）123：1053）． 40－kDaOMPisconservedamongandexpressedbymanyP gingivalisstrains（Hiratsuka，K．et．al．，1996，FEMS．Micro－ biol．Lett．138：167－172）．Amonoclonalantibodyagainstthe 40－kDaOMPantlgeninhibitsthecoaggregationofPgin－ glValisandActinomycesviscosus．Theantlgenisanimpor－ tantfactorforcolonizationofPgingivalis（Abiko，Yetal．， Infect．hlmun．，1997Ⅵ）165：3966；Hiratsuka，K．et al．， Arch．Oral．Biol．，1992Ⅵ）137：717；Saito S．et al．，Gen． Pharmacol．，1997Ⅵ）128：675）．Ithasalsobeenreportedthat ananti－40－kDa OMPpolyclonalantibody has activityof PrOmOtingthephagocyticabilityofapromyelocyticcellline HL60（Saito，S．etal．，J．Periodontol．，1999Ⅵ）170：610）． Hence，1tis agreatcontributiontothe development ofa noveltherapeuticmethodforperiodontaldiseasestoprovide ahumanmonoclonalantibodythathasactivityofinhibiting thecoaggr甲ationofPgingivalisandactivityofpromoting PhagocytosIS OfP ginglValis byleukocytes and thatis excellentintermSOfsafetyandlong－1astlnge正己ctswhenit is appliedto patients．However，nO SuChantibodies have beenreported．Ingeneral，1tisnotalwaysclearwhetherall antibodieshavingactivltyOfinhibitingthecoaggregationof P ginglValis have activlty Ofpromotlng Steri1ization by leukocytes・FurthermOre，itisknownthatincorporation

．

OfthepathogenicityofP gingivalis．Inthe40－kDaOMP PrOteln，ahemeregulatorymotifthatisknownasahemin－ binding siteis present．Ithas beenactuallyreportedthat 40－kDaOMPisatypeofhemin－bindingprotein（Shibata，Y et al．，B．B．R．C．，2003Ⅵ）1300：351）．Anantibody that inhibitsthebindingof40－kDaOMPtoheminislikelyto inhibit the growth and prolifbration ofP ginglValis and StrOnglydamageSPginglValis．

TIIERAPEUTIC AGENT FOR PERIODONTAL DISEASE

TECHNICAL FIELD

［0001］Thepresentinventionrelatestoanagentthatacts agalnStPeriodontaldiseases．Specifically，thepresentinven－ tionrelatestoahumanmonoclonalantibodyagalnSt40－kDa OMIlthathasbothactivltyOfinhibitingtheaggregationof Pathogenic bacteriainvoIvedin periodontaldiseases and activltyOfpromotlngSterilizationbyleukocytes，thatpref－ erablyinhibitsthebindingof40－kDaOMPtohemin，and that has alower possibilityofcausing side e正己cts than antibodies derivedfromnon－humananimals such as mice．

The present invention also relates to an agent that acts against periodontal diseases containing such monoclonal antibody．

BACKGROUNDART

［0002］Periodontaldiseases affect about80％or more PeOPleinJapan．Majorcausesofperiodontaldiseasesare thoughttobeinfbctionwithoralbacteria，increas？Sin PathogenicbacteriainvoIvedinperiodontaldiseases，1nVa－ Sion oftissues by bactena，immune reSPOnSeSin hosts agalnStinfbction，andthelike．Periodontaldiseasesfinally CauSelossofteethandthusareverylmPOrtantdiseasesthat CauSethequalityoflifbtodeteriorate．Recently，Periodontal diseaseshavebeenshowntohavecausalrelationshipsnot Only withtoothloss，but also withcirculatory disorders， deliveryofunderweightbabiesandearlydelivery，diabetes， endocarditis，and pneumonia（Abiko Y，Crit．Rev．Oral． Biol．Med．，2000Ⅵ）111：140）．

［0003］In therapeutic methodsfor periodontaldiseases， eliminationoftheinfectionsourceistherapeuticallylmPOr－ tant．However，eXtremely pnmitive methodsincluding bruShingandscaling，Periodontalsurgery，andthelikeare Stillm叫Ortherapeuticmethodsforperiodontaldiseases．Itis impossible to eradicate pathogenic bacteriainvoIvedin Periodontaldiseases with only such therapleSinvoIving mechanicalelimination．FurthermOre，1tmaybeimpossible to apply suchtherapleStOPatientswithsystemic disease， because bacillaemia or focalinfbctionis alsoinduced． FurthermOre，administrationofantibiotics（e．g．，tetraCyCline andminocycline）orantibacterialagentshasbeenconsidered tobeeffectiveperiodontaltherapy．However，CurrentPrOb－ 1emsincluding multipletypes ofresistantbactena，reSis－ tance－related genes，Side e正己cts，and thelike have been indicated．Limitation ofpharmaCOtherapy has also been SuggeSted．Hence，in the present situation，nO effective therapeuticmethodshavebeenestablishedagalnStPeriodon－ taldiseases．Thedevelopmentofanewtherapeuticmethod thatisextremelysafbhrhumanbodiesandenableseradi－ CationofpathogenicbacteriainvoIvedinperiodontaldis－ easeshasbeenawaited．

［0004］Oforalbacteria，Po77，毎7VmOnaSgfngivalis（P

gingivalis）hasbeenisolatedathighfrequencleSfromthe Periodontalpockets of adult－type periodontaldisease Patients．P ginglValisis thus the strongest suspect as a PathogenicbacteriainvoIvedinperiodontaldiseases．This bacteriumis a gram－negative bacillus thatformS black COloniesonabloodplate．Itisthoughtthatthesurfhcelayer COmPOnentSOfthebacterialbodyandextracellularproducts areresponsiblefordisruPtlOnOfperiodontaltissues．Hence，

US2007／0036734Al Feb．15，2007

［0016］［7］an antibody binding to40－KDa OMP or a functionalfragmentthereof，Whichhas activltyOfsup－ PreSSlngalveolarboneresorptlOn；

［0017］［8］theantibodyorthefunctionalfragmentthereof チCCOrdingtoanyoneof［1］to［7］，Whereintheantibody lSahumanantibody；

［0018］［9］theantibodyorthefunctionalfragmentthereof accordingtoanyoneof［1］to［8］，Whichisproducedby amouse－mOuSehybridoma；

［0019］［10］theantibodyorthefunctionalfragmentthereof チCCOrdingtoanyoneof［1］to［9］，Whereintheantibody

lSamOnOClonalantibody；

［0020］［11］theantibodyorthefunctionalfragmentthereof accordingtoanyoneof［1］to［10］，Whichcovalentlyor non－COValentlybindstoatherapeuticagent；

［0021］［12］theantibodyorthefunctionalfrag㌣entthereof

according to［11］，Wherein the therapeutlC agentis Selectedfromantibioticsorantibacterialagents；

［0022］［13］theantibodyorthefunctionalfragmentthereof accordingto［12］，Whereintheantibioticortheantibac－ terialagentistetracyclineorminocycline；

［0023］［14］theantibodyorthefunctionalfragmentthereof accordingtoanyoneof［1］to［13］，Whereintheantibody ClassisIgG；

［0024］［15］theantibodyorthefunctionalfragmentthereof accordingto［14］，WhereinIgGisIgGl；

［0025］［16］theantibodyorthefunctionalfragmentthereof accordingtoanyoneof［1］to［13］，Whereintheantibody ClassisIgA；

［0026］［17］The antibody or thefunctionalfragment thereofaccordingtoanyoneof［1］to［16］，Whereinthe aminoacidsequenceofaheavychainconstantreglOnis altered；

［0027］［18］an antibody binding to40－kDa OMP or a functionalfragment thereof，Whichis produced by a hybridomah13－17（accessionNo．FERMBP－8325）；

［0028］［19］an antibody binding to40－kDa OMP or a functionalfragment thereof，Which comprlSeS Variable reglOnSOfanantibodythatisproducedbyahybridoma h13－17（accessionNo．FERMBP－8325）；

［0029］［20］theantibodyorthefunctionalfragmentthereof according to［18］or［19］，Which covalently or non－ COValentlybindstoatherapeuticagent；

［0030］［21］theantibodyorthefunctionalfrag㌣entthereof





DISCLOSUREOFTHEITWENTION

［0007］AnobjecfOfthepresentinventionistoprovidean agentthatactsagalnStPeriodontaldiseases，Whichissafefor human bodies while eliminatlng P ginglValis，Whichis knownas apathogenicbacteriuminvoIvedinperiodontal diseases，fromwithintheperiodontalpocket．Particularly，an Object of the presentinventionis to provide a human monoclonalantibody．Suchhumanmonoclonalantibodyhas both activityofinhibiting the aggregation ofthe above Pathogenic bacteriainvoIvedin periodontaldiseases and activltyOfpromotlngSterilizationbyleukocytes，1SPrefer－ ably amonoclonalantibody agalnSt40－kDa OMIミwhich

inhibitsthebindingofhemin，andfurther，haslowerpossi－ bilityofcausipgsidee正己ctsthanantibodiesderivedfrom non－humananlmals suchas mice．

［0008］Anobjectofthepresentinventionistoachievethe aboveconventionalobjects．Specifically，Wehaveconducted intensive studies to develop an agent that acts agalnSt Periodontaldiseases，Whichissafbhrhumanbodieswhile eliminatlng P ginglValis，Whichisknown as pathogenic bacteriainvoIvedinperiodontaldiseases，fromwithinthe Periodontalpocket．Asaresult，Wehavediscoveredthata humanmonoclonalantibodyhavingbothactivityofinhib－ itlngtheaggregationofthepathogenicbacteriainvoIvedin Periodontaldiseasesandactivityofpromotingsterilization byleukocytes，being prefbrably a monoclonalantibody agalnSt40－kDaOMIミwhichinhibitsthebindingofhemin，

andfurther，havinglowerpossibilityofcausi？gSidee正己cts than antibodies derivedfrom non－human anlmals such as

micehasgoodeffectsasanagentthatactsagalnStPeriodon－ taldiseases．Thus，Wehavecompletedthepresentinvention．

［0009］Thepresentinventionisasfbllows：

［0010］［1］an antibody binding to40－kDa OMP or a functionalfragmentthereof，Whichhasactivityofinhib－ itlngthebindingofheminto40－kDaOMP；

［0011］［2］an antibody binding to40－kDa OMP or a functionalfragment thereof，Which has（1）activity of inhibitingthe co？ggregationofP gingivalis and（2） activityofpromotlnghumanneutrophilicphagocytosis；

［0012］［3］an antibody binding to40－kDa OMP or a functionalfragment thereof，Which has（1）activity of inhibiting the coaggregation of P gingivalis and（2） activityofinhibiting the binding ofhemin to40－kDa OMP；

［0013］［4］an antibody binding to40－kDa OMP or a functionalfragment thereof，Which has（1）activity of Pr〔TPOting human neutrophilic phagocytosis and（2） actlVityofinhibiting the binding ofhemin to40－kDa OMP；

［0014］［5］an antibody binding to40－kDa OMP or a functionalfragment thereof，Which has（1）activity of inhibitingthecoaggregationofPgingivalis，（2）activity Ofp？mOtinghumanneutrophilicphagocytosis，and（3） activltyofinhibiting the binding ofhemin to40－kDa OMP；

［0015］［6］theantibodyorthefunctionalfragmentthereof accordingfOanyOneOf［2］，［3］，and［5］，Whereinthe COaggregatlOn OfP glnglValisis coaggregation ofP gJ〝gJVdJ∫∫andノ4c～∫〝0〝ぴCe∫V∫∫CO∫〟∫ノ・

US2007／0036734Al Feb．15，2007


［0035］［26］theantibodyorthefunctionalfragmentthereof accordingtoanyoneof［18］to［25］，Whereintheamino acidsequenceofaheavychainconstantreglOnisaltered；

［0036］［27］a hybridoma h13－17（accessionNo．FERM BP－8325）；

［0037］［28］an antibody binding to40－kDa OMP or a functionalfragment thereof，Whichis produced by a hybridoma5－89－2（accessionNo．FERMBP－8323）；

［0038］［29］an antibody binding to40－kDa OMP or a functionalfragment thereof，Which comprlSeS Variable reglOnSOfanantibodythatisproducedbyahybridoma 5－89－2（accessionNo．FERMBP－8323）；


［0040］［31］theantibodyorthefunctionalfragヮentthereof







［0046］［37］a hybridoma5－89－2（accessionNo．FERM BP－8323）；

［0047］［38］an antibody binding to40－kDa OMP or a functionalfragment thereof，Whichis produced by a hybridomaa44－1（accessionNo．FERMBP－8324）；

［0048］［39］an antibody binding to40－kDa OMP or a functionalfragment thereof，Which comprlSeS Variable reglOnSOfanantibodythatisproducedbyahybridoma a44－1（accessionNo．FERMBP－8324）；


［0050］［41］theantibodyorthefunctionalfragmentthereof accordingto［40］，Whereinthetherapeuticagentisanti－ bioticsorantibacterialagents；






［0056］［47］a hybridoma a44－1（accession No．FERM BP－8324）；

［0057］［48］anucleicacid，Whichispo ．

h13－17（accessionNo．FERM BP－8325），a hybridoma 5－89－2（accessionNo．FERMBP－8323），andahybridoma a44－1（accessionNo．FERMBP－8324）andencodes an antibody containlng a Variable reglOn Of an antibody PrOducedbytheabovehybridomaorafunctionalfrag－ mentofthesaidantibody；

［0058］［49］aproteinencodedbythenucleicacidaccord－ ingto［48］，Whichisanantibodyorafunctionalfragment thereof；

［0059］［50］anexpressionvector，Whichhasthenucleic acidaccordingto［48］；

［0060］［51］a host，Which has the expression vector accordingto［50］；

［0061］［52］thehost

．

insectcells，manlmaliancells，Plantcells，andmanlmals；

［0062］［53］amethodforproducingチnantibodybindingto 40－kDa OMIミwhich comprlSeSISOlatlng a gene that

encodes an antibody binding to40－kDa OMPfrom a hybridomaselectedfromthegroupconsistlngOfahybri－ domah13－17（accessipnNo・FERMBP－8325），ahybri－ doma5－89－2（accesslOn No．FERM BP－8323），and a hybridomaa44－1（accessionNo．FERMBP－8324），COn－ StruCtlnganeXPreSSionvectorcompnslngthegene，1ntrO－ ducingtheexpressionvectorintoahosttocauseexpres－ Sionoftheantibody，andcollectlngtheantibodyfromthe Obtained host，the culture supernatant ofthe host，Or SeCretionfromthehost；

［0063］［54］anag？ntforsuppressingalveolarboneresorp－ tion，WhichcontalnSanantibodybindingto40－KDaOMP Orafunctionalfragmentthereofasanactivelngredient；

［0064］［55］anagentforpreventing，diagnosing，Ortreat－ 1ng Periodontaldiseases，Which contains an antibody bindingto40－kDaOMPorafunctionalfragmentthereof asanactivelngredient；

［0065］［56］useofanantibodybindingto40－KDaOMPor afunctionalfragmentthereofforproductionofanagent hrsuppresslngalveolarboneresorptlOn；

［0066］［57］a method fbr suppr竺SSing alveolar bone

resorptlOn，Whichcompnsesprepannganantibodybind－ 1ngtO40－KDaOMPorafunctionalfragmentthereofand administenngtheantibodyorthefragmenttoananimal；

US2007／0036734Al Feb．15，2007

［0067］［58］useofanantibodybindingto40－KDaOMPor afunctionalfragmentthereofforproductionofanagent forpreventlng，diagnoslng，Ortreatlng Periodontaldis－ eaSeS；

［0068］［59］amethodfordiagnosing，Pr？Venting，0てtreat－ 1ngPeriodontaldiseases，WhichcomprlSeSPrePanngan antibodybindingto40－KDaOMPorafunctionalfrag－ mentthereofandadministerlngtheantibodyorthefrag－ menttoananimal；

［0069］［60］anagentforpreventing，diagnosing，Ortreat－ 1ngPeriodontaldiseases，Whichcontainstheantibodyor thefunctionalfragmentthereofaccordingtoanyoneof ［1］to［26］，［28］to［36］，［38］to［46］，and［49］asanactive

lngredient；

［0070］［61］anageptforsuppressingalveolarboneresorp－

tion，WhichcontalnStheantibodyorthefunctionalfrag－ mentthereofaccordingtoanyoneof［1］to［26］，［28］to ［36］，［38］to［46］，and［49］asanactiveingredient；

［0071］［62］useoftheantibodyorthefunctionalfragment thereofaccordingtoanyoneof［1］to［26］，［28］to［36］，

［38］to［46］，and［49］hr production ofan agentfor PreVentlng，diagnoslng，OrtreatlngPeriodontaldiseases；

［0072］［63］useoftheantibodyorthefunctionalfragment thereofaccordingtoanyoneof［1］to［26］，［28］to［36］，

［38］to［46］，and［49］hr production ofan agentfor SuPPreSSlngalveolarboneresorptlOn；

［0073］［64］amethodfordiagnosing，PrPVenting，Oftreat－

1ngPeriodontaldiseases，Whichcompnsespreparlngthe antibodyorthefunctionalfragmentthereofaccordingto anyoneof［1］to［26］，［28］to［36］，［38］to［46］，and［49］ and administering the antibody or the fragment to an animal；and

［0074］［65］a methodfor suppr？SSing alveolar bone

resorptlOn，WhichcomprlSeSPreParlngtheantibodyorthe functionalfragmentthereofaccordingtoanyoneof［1］to ［26］，［28］to［36］，［38］to［46］，and［49］andadministering

theantibodyorthefragmenttoananimal．

［0075］Thisdescriptio？includespartOrallofthecontents

asdisclosedinthedescnptlOnand／ordrawingsofJapanese PatentApplicationNo．2003－072714，Whichis a pnorlty documentofthepresentapplication．

BRIEF DESCRIPTION OF THE DRAWINGS

［0076］FIG．1shows the reactivityofeach anti－40kDa OMPantibodyagalnStr40－kDaOMPandPginglValis．

［0077］FIG．2showsacomparisonofthebindingactivity Ofeach anti－40－kDa OMP antibody and the serum Ofa PeriodontaldiseasepatienttoPginglValis．

［0078］FIG．3shows the results ofanalyzing reactions betweeneachanti－40－kDaOMPantibodyandPginglValis．

［0079］FIG．4showsthee正己ctofeachanti－40－kDaOMP antibody on theinteraction between40－kDa OMP and hemin．

［0080］FIG．5shows an h13－17antibody’s activity of inhibitingthebindingof40－kDaOMPtohemin．

［0081］FIG・6sho㌣Sthereactivityofeachanti－40－kDa

OMPantibodytovanousPginglValisstrains．

［0082］FIG．7shows eachanti－40－kDaOMPantibody’s activityofsuppressingratalveolarboneresorption．

BEST MODE OF CARRYING OUT THE

ITWENTION

［0083］Thepresentinventionwillbedescribedindetai1as 払1lows．

［0084］40－kDa OMP can be produced by appropriate employmentofamethodknownintheteclmicalfield，SuCh asachemicalsynthesismethodoracellculturemethod，in addition to a gene recombination teclmique based on a known nucleotide sequence（DNA Data Bank ofJapan： accessionNo．ABO59658）oraknownaminoacidsequence． Thenucleotideseque？CeOf40－kDaOMPisshowninSEQ IDNO：1andtheam1nOaCidsequenceof40－kDaOMPis ShowninSEQIDNO：2．Furthermore，aPartialsequenceof 40－kDaOMPcanalsobeproducedaccordingtoamethod knownintheteclmicalfieldanddescribedlater，SuChasa gene recombination technique or a chemical synthesis method．40－kDa OMP can be produced by appropnate CleavageuslngPrOteinaseorthelike．

［0085］Theantibodyorthefunctionalfragmentthereofof the presentinvention encompasses various anti－40－kDa OMPmonoclonalantibodiesorfunctionalfragmentsthereof havingthefbllowingreactivities．Specifically，（1）ananti－ body binding to40－kDa OMP or afunctionalfragment thereofhavingactivltyOfinhibitingthecoaggregationofP gingivalis and activityofpromoting human neutrophilic Phag？CytOSis，（2）anantibodybindingto40－kDaOMPora functlOnalfragmentthereofhavingactivltyOfinhibitingthe COaggregationofPginglValis，aCtivltyOfpromotlnghuman neutrophilic phagocytosis，and activityofinhibiting the bindingofheminto40－kDaOMIl（3）anantibodybinding to40－kDa OMPor afunctionalfragmentthereofhaving activityofpromotinghumanneutrophilicphagocytosisand activityofinhibitingthebindingofheminto40－kDaOMIl （4）an antibody bindingto40－kDa OMP or afunctional fragmentthereofhavingactivltyOfinhibitingthecoaggre－ gationofPgingivalisandactivityofinhibitingthebinding Ofheminto40－kDaOMIミor（5）anantibodybindingto

40－kDaOMPorafunctionalfragmentthereofhavingactiv－ 1tyOfinhibitingthebindingofheminto40－kDaOMPare encompassed．

［0086］”Coaggregation of P gingivalis”indicates the aggregationofPginglValiswithothermicroorganismssuch asActinonv）CeSViscosusand5わ甲tOCOCCuSgO7donii．Such aggregationresultsincolonizationofpathogenicbacteriain theformOfplaquesintheperiodontalpocket．Accordingly， “activityofinhibitingtheaggregationofPgingivalis”ofthe antibodyofthepresentinventionindicatesactivitybywhich aggregation ofP glnglValis with other bacteria can be inhibited．WhetherornotanantibodyhassuchactivltyOf inhibiting aggregation can be determined by a method describedinExample9inthisdescription．Whenactivityof inhibitingaggregationismeasuredbythemethoddescribed in ExamPle9，the score ofthe antibody ofthe present inventionisprefbrably20rless．FurthermOre，”activityof PrOmOtingsteri1izationbyleukocytes”oftheantibodyofthe PreSentinventionindicatesactivltyOfpromotlngthephago－ CytOSisofP gingivalisbyleukocytessuchasneutrophils． Suchactivitycanbedeterminedbyamethoddescribedin ExamPlelOin this description．When such activityof

US2007／0036734Al Feb．15，2007

promoting phagocytosis is measured by the method describedinExamplelO，thephagocytosisrateinthecase Ofthe antibody ofthe presentinventionis slgnificantly higherthanthatinthecaseofacontrolantibody．Further－ more，theantibodyofthepresentinventionencompassesan antibody having activlty Ofinhibiting the binding ofP glnglValis40－kDa OMP to hemin．Whether or not such antibodyinhibitsthebindingofPginglValis40－kDaOMP to hemin canbe determinedbythe method describedin ExamPle14in this description．When such activityof inhibitingthebindingtoheminismeasuredbythemethod describedinExamPle14，theactivityoftheantibodyofthe PreSentinventionisslgnificantlyhigherthanthatofacontrol antibody．PginglValisisusedforverificationoftheactivlty Oftheantibodyofthepresentinvention．AnyPginglValis maybeused，aSlongasitexpresses40－kDaOMP．Examples OfsuchPginglValisincludeP ginglValis381usedinthe ExamPles ofthepresentinventionandP ginglValisW50 （ArCCNo．53978）．

［0087］ExamPlesofsuchantibodyorafunctionalfra竿－ mentthereofincludeananti－40－kDaOMPmonoclonalantl－ bodydescribedlaterandamonoclonalantibodycompnslng aheavychainand／oralightchainhavinganamino acid SequenCederivedfromtheaminoacidsequenceofaheavy Chain and／or alight chain composlng the antibody by deletion，Substitution，Oradditionoflorseveralaminoacids andhavingthereactivitya9COrdingtoa？yOI誓Of（1）to（5） above．“Alteration”（deletlOn，SubstitutlOn，1nSertion，and addition）ofaminoacidsasdescribedabovecanbeintro－ ducedbypartiallyaltenngthenucleotidesequenceencoding arelevantaminoacidsequence．ForexamPle，SuChpartial alterationinanucleotidesequencecanbeintroducedbya Standard method uslngknown site－SPeCific mutagenesis （ProcNatlAcadSciU．S．A．，198481：5662；Sambrooketal．， Molecular CloningA Laboratory Manual（1989）Second edition，Cold Spring Harbor Laboratory Press）．For example，anantibodywhereintheaminoacidsequenceofa heavychainconstantreglOnhasbeenalteredinthepresent inventionmayhavestrongeractivityofpromotingphago－ CytOSis ofP gingivalis byleukocytes than that of the antibodybeforealterationbecauseofimproveda丑inityfor anFcreceptor．

［0088］”Antibody”ofthepresFntinventionalsoencom－ PaSSeS anantibodyhavinganylnlmunOglobulinclassand Subclassandispreferablyanantibodyhavinghumaninlmu－ noglobulin class and subclass．Examples of preferable Classes（andsubclasses）areinlmunOglobulinG（IgG）and IgA，andparticularlyIgGlandIgA．

［0089］Anotherpreferableexampleoftheantibodyorthe fragment thereofofthe presentinventionis a sequence COmPOSlngamOnOClonalantibodyorafragmentthereof， WhichrecognlZeSanePltOPeintheaminoacidsequenceof 40－kDaOMPandhasthereactivityaccordingtoanyoneof （1）to（5）above．

［0090］Afragmentofanantibodyinthepresentinvention meansaportionoftheantibodyasdefinedabove．Specific examplesofsuchfragmentincludeF（abT）2，Fab’，Fab，Fv， disulphide－1inkedFV Single－ChainFV（scFV），andpoly－ mersthereof（D．J．King．，ApplicationsandEngineeringof MonoclonalAntibodies．，1998T．J．InternationalLtd）．Such antibody fragment can be obtained by a conventional method，includingdigestionofanantibodymoleculewith

PrOteaSe SuChaspapalnOrPePSlnOrbyaknowngenetic englneerlngteClmique．“FunCtionalfragment”meansafrag－ mentofanantibodythatspecificallybindstoanantlgentO Whichacompleteantibodyspecificallybinds．

［0091］Theantibodyofthepresentinventioncanbepro－ ducedbythefbllowlngmethod，forexample．Specifically， forexamPle，anOn－hum禦mammal（e・g・，amOuSe，arabbit， agoat，Orahorse）isチnlmunizedwith40－kDaOMPas definedaboveoraportlOnthereof，aPrOductobtainedby bindingwithanappropriatesubstance（e．g．，bovineserum albumin）hrenhancingtheantigenicityofanantigen，Ora Cellexpresslng40－kDaOMPonthecellsurfacesinlarge quantities，tOgetherwithaninlmunO－augmentingagent（e．g．， Freund’s aqjuvant），ifnecessary．Alternatively，aneXPreS－ Sionvectorintowhich40－kDaOMPhasbeenincorporated isadministeredtoanon－humanmanlmal，SOthatinlmuni－ Zationcanbe carriedout．Amonoclonalantibodyispro－ ducedbyprepanngahybridomaofanantibody－PrOducing cellobtainedfrom aninlmunized animaland a cellof a

myelomacellline（myelomacell）incapableofproducing anyautoantibody，Clonlngthehybridoma，andthenselectlng Clonesthatproducemonoclonalantibodiesshowingspecific a伍nityforanantlgenuSedforinlmunization．Moreprefbr－ ably，throughtheuseofanon－humananimalthatretainsan unrearrangedhumanantibodygeneandproducesahuman antibodyspecifictotherelevantinlmunOgenaSareSultof inlmunization，theantibodyofthepresentinventioncanbe Obtainedas ahumanantibody．AnexamPle ofsuchnon－ humananimalisamouse．Amethodfbrproducingamouse CaPable ofproducing a human antibodyis describedin InternationalPatentPublicationWOO2／43478．Here，”human antibody”meansanantibodythatisanexpressionproduct Ofahuman－derivedantibody geneormeans afunctional fragmentthereof二Examplesofthemonoclonalantibodyof thepresentinventionincludemonoclonalantibodies pro－ ducedbyhybridomaclonesh13－17（accessionNo．FERM BP－8325），5－89－2（FERM BP－8323），and a44－1（FERM BP－8324）thatwereinternationallydepositedattheInterna－ tionalPatentOrganismDepositaryoftheNationalInstitute OfAdvancedIndustrialScienceandTeclm0logy（Tsukuba Centra16，1－1－1Higashi，Tsukuba，Ibaraki，Japan）asofMar． 11，2003，undertheBudapestTreaty，Orfunctionalfragments thereof．Moreover，antibodiesobtainedbyalterationofthe classesorsubclassesofthesemonoclonalantibodiesarealso included．FurthermOre，antibodiesobtainedbyalterlngthe aminoacidsequencesoftheheavychainconstantreglOnSOf antibodies produced by these hybridomas or functional fragmentsthereofarealsoincluded．

［0092］Thepresentinventionalsoencompassesanucleic acidthatispossessedbytheabovehybridomaandencodes an antibody containing a variable region of an antibody PrOducedbythehybridomaandanucleicacidencodinga functionalfragmentofsuchantibody．Thesenucleicacids CanbeobtainedbyageneralgeneticenglneerlngteClmique fromhybridomas．Thenucleotidesequencesthereofcanalso bedeterminedbyaknownmethodfordeterminlngnuCle－ Otide sequences．Furthermore，the presentinvention also includes proteins encoded by nucleic acids obtained as describedabove．Suchproteinshavereactivityaccordingto anyoneof（1）to（5）above・Moreover，thepreヲentinvention

alsoencompassesexpressionvectorscontainlngtheabove nucleic acids and host cells containlng SuCh expression vectors．Such vectors and host cells are notlimited．

ExamPlesofsuchhostsincludenotonlyEscherichiacoli，

US2007／0036734Al Feb．15，2007

yeastcells，1nSeCtCells，manlmaliancells，andplantcells，but alsoincludeinsectindividuals andmammalianindividuals．

ExamPles ofinsectindividualsinclude silkwormS and examples of manlmalianindividualsinclude mice，ratS， Cattle，horses，Sheep，andpigs，butarenotlimitedthereto．As expressionvectors，knownvectorsandconlmerCialvectors COrreSPOndingtoeachhostcanbeused．FurthermOre，1nSeCt individualsormanlmalianindividualscanbetransfbctedby known methods uslng eXPreSSion vectors．The present inventionfurtherencompassesamethodforproducingthe antibodyorthefunctionalfragmentthereofofthepresent invention，Which comprlSeS CauSlng eXPreSSion of the nucleicacidofthepresentinventioninhostcellsorhost individualsthatcontainanexpressionvectorcontainlngthe nucleicacidandcollectlngtheantibodyorthefunctional fragmentthereof（expressionproduct）fromtheculturesolu－ tionofthehostcells，ahostbodyfluid，Orahostsecretion suchas milk．

［0093］Specifically，the antibody orthefunctionalfrag－ mentthereofofthepresentinventioncanbeproducedas hllows．AhybridomasecretlngamOnOClonalantibodycan bepreparedbythemethodofKohlerandMilsteinetal．（Nature．，1975Ⅵ）1．256：495－497），Or aCCOrding to such method．Specifically，antibody－PrOducingcellsarecontained inthespleen，1ymphnode，bonemarrow，tOnSil，Orthelike， OrPrefbrablythelymphnodeorthespleenobtainedfroman animalinlmunizedasdescribedabove．Suchantibody－PrO－ ducingcellsarefusedwithmyelomacellsthatarederived fromamanlmalandprefbrablyfromamouse，rat，gulnea Pig，hamster，rabbit，human，Orthelikeandareincapableof PrOducing any autoantibody．Thus，the antibody or the functionalfragmentthereofofthepresentinventioncanbe PrePared．Forexample，Cellfusioncanbe carriedoutby mixlng antibody－PrOducingcellswithniyelomacellsina high－COnCentrationpolymersolutionofpolyethyleneglycol Orthelike（e．g．，Withamolecularweightrangingfrom1500 to6000）generallyatatemperaturヲrangingfromapproxi－ mately300C．to400C．hrapproxlmatelyltolOminutes． Screenlngfbrhybridomaclonesproducingmonoclonalanti－ bodiescanbecarriedoutasfbllows．ForexamPle，hybri－ domas are culturedonamicrotiterplate．Reactivityofa Culturesupernatantfromwellinwhichtheproliferationof thehybridomahasbeenobsetbedtoanantlgenforinlmu－ nizationismeasuredusinganinlmunOloglCalmethodsuch astheenzyme－1inkedinlmunOaSSay（e．g．，ELISA），radioim－ munoassay，Orfluorescentantibodyteclmique．

［0094］Amonoclonalantibodycanbeproducedfr0IPa hybridomabycultunnglnVitrohybridomasandthenlSO－ 1atlng mOnOClonalantibodiesfrom a culture supernatant． FurthermOre，hybridomas canbe culturedin vivoin the asciteorthelikeofamouse，rat，gulneaPig，hamster，rabbit， orthelike andthenthemonoclonalantibodies canalso be isolatedfromthe ascite．

［0095］FurthermOre，areCOmbinantantibodycanbepre－ Paredbya generecombinationteclmique，SPeCificallyby Clonlnga gene enCOdingamonoclonalantibodyfroman antibody－PrOducingcellsuchasahybridoma，1nCOrPOratlng theresultantintoanappropnatevector，andthenintroducing thevectorintoahost（e．g．，aCellofamanlmaliancellline SuChasaChinesehamSterOVary（CHO）cell，Egcherichia COli，yeaSt Cell，insect cell，OrPlant cells）（RJ．Delves．， ANTIBODY PRODUCTION ESSENTIAL TECH－

NIQUES．，1997WILEYP．ShepherdandC．Dean．，Mono－

ClonalAntibodies．，20000XFORDUNIVERSITYPRESS， J．W．Goding．，MonoclonalAntibodies：PrlnCiplesandprac－ tice．，1993ACADEMIC PRESS）．Moreover，tranSgenic Cattle，gOatS，Sheep，OrPlgSWhereinatargetantibodygene isincorporatedin an endogenous gene can be produced uslngatranSgenicanimalproductionteclmique．Monoclonal antibodies derived from the antibody gene can also be Obtainedinlargequantitiesfromthemilkofthetransgenic animals．Hybridomascanbeculturedinvitroasfo1lows．In accordancewithvariousconditionsincludingtheproperties Ofcellspecies to be cultured，the purposes oftests and Studies，Culture methods，and thelike，known nutrition mediaoreverynutritionmediathatareinducedandpre－ Paredfromknownbasicmediaareusedfbrculture．Such media enable proliferation，maintenance，and storage of hybridomas and are used for causing the hybridomas to PrOducemonoclonalantibodiesinculturesupernatantS．

［0096］Thethusproducedmonoclonalantibodiescanbe Purifiedbyappropnatecombinationofmethodsknownin theart，SuChaschromatographyuslngaPrOteinAcolumnor PrOtein G colunln，10n eXChange chromatography，hydro－ Phobic chromatography，anammOniumsulfatesaltingout method，gelfi1tration，andaBinitychromatography．

［0097］Themonoclonalantibodyorthefragmentthereof Ofthepresentinventionproducedbytheabovemethodcan beusedtoformaCOmPlex．Suchcomplexcanbeusedfbr treatmentsuchasmissiletherapybycoIuugatlngtheanti－ body or thefragment thereof to a drugfor treatment． ExamPlesofsuchdrugfortreatmenttobecoIuugatedtoan antibodyinclude，butarenotlimitedto，antibioticssuchas tetracycline andminocycline andantibacterialagents．An antibody may be coruugatedto a drug fbrtreatment via eithercovalentornon－COValentbonding（e・g・，50nicbond－ ing）．Forexample，areaCtivegroup（e．g．，anamlnOgrOuP，a Carboxylgroup，and a hydroxylgroup）in an antibody moleculeoracoordinationgroupISuSed．Specifically，the COmPlexofthepresentinventioncanbeobtainedbycauslng anantibodytocomeintocontactwithadrugfbrtreatment COntainingafunctionalgてOuP（inthecaseofbacterialtoxin andachemicaltherapeutlCa声ent）capableofreactingwith thereactive草rOuPOrCOntainlnganioni？grOuP（inthecase OfaradioactlVenuClide）capableofform1ngaCOmPlexwith thecoordinationgroup．Alternatively，abiotin－aVidinsystem Canalsobeusedfbrtheformationofsuchcomplex．Fur－ thermOre，Whenadrugfortreatmentisaproteinorapeptide， thecomplexofthepresentinventioncanalsobeproduced asafusionproteinofanantibodywiththeaboveproteinor PePtidebyageneticenglneerlngteClmique．

［0098］Moreover，theanti－40－kDaOMPantibodyofthe PreSentinventionorapharmaCeuticalcompositionfbrpre－ Ventlng，diagnoslng，OrtreatlngPeriodontaldiseases，Which COntains the anti－40－kDa OMP antibody coI勺ugatedto a drug fbr treatment as described above，1S alsoincluded Withinthescopeofthepresentinvention．Throughadmin－ istrationoftheanti－40－kDaOMPantibodyofthepresent invention，P ginglValis can be eliminatedfrom the oral CaVlty．FurthermOre，disruPtlOnOfperiodontaltissuesdueto PginglValiscanbepreventedandperiodontaldiseasescan bepreventedandtreated．Suchcompositionshouldcontain thetherapeuticallye正己ctivedoseofadrugfortreatmentand is fbrmulatedinto various fbrmS fbr oraland parenteral administration．Here，”therapeuticallye正己ctivedose”indi－

CateSanamOuntOfthecompositionthatcanhavetherapeutic

US2007／0036734Al Feb．15，2007

e正己ctsonglVenSymPtOmSOrCanimproveadministration Plans．Thecompositionofthepresentinventioncancontaln， inadditiontoanantibody，1typeorapluralityoftypesof PhysiologlCallyacceptablepharmaceuticaladditivessuchas adiluent，aPreServative，aSOlubilizlngagent，anemulsifier， anaqjuvant，anantioxidant，anisotonizlngagent，aneXCIPl－ ent，and a carrier．Furthermore，the composition ofthe PreSentinventionmayalsobepreparedaspartofamixture WithanotherantibodyoranotherdrugSuChasanantibiotic． ExamPles ofan approprlate Carrierinclude，but are not limitedto，aPhysiologlCalsalinesolution，Phosphatebuff－ ered saline，aPhosphatebufferedsaline glucose solution， andabu正己redsalinesolution．FurthermOre，thecomposition mayalsocontainastabilizer，SuChasaminoacids，SugarS，a Surfhctant，Or an agent fbr preventlng adsorptlOn tO the Surfhce，Whichareknownintheart．Examplesofsuchform OfapharmaCeuticalpreparationincludeapaste，aliquid，a freeze－driedpharmaceuticalpreparation（inthis case，the PreParationcanbereshapedbytheadditionofabuffered aqueous solution as described above and then used），a SuStained release pharmaceuticalpreparation，an enteric COatedpharmaceuticalpreparation，anlrUeCtion，anddrops． TheformOfpharmaCeuticalpreparation can be selected fromtheseexamplesaccordingtotreatmentpurposesand treatmentplans．

［0099］PossibleroutesofadministratioareOraladminis－ tration and non－enteraladministrationlnCludinglntraVe－ nous，intramuscular，Subcutaneous，andintraperitoneal lI勺eCtions ordrugdelivery．AnoptlmumrOuteis selected basedontestsuslnganimals．Alternatively，anOtherpossible methodinvoIves causlng the composition ofthe present inventiontocomeintodirectcontactwithanaffectedreglOn Ofapatient．Inviewofdirectapplicationofthecomposition toareglOna正己ctedbyperiodontaldiseases，1tispreferable to administerthe compositioninto the oralcavityorthe Periodontalpocket．Thedoseisapproprlatelydeterminedby implementationoftestsuslnganimalsandclinicaltests．In general，COnditionsorseverity，age，bodyweight，SeX，and thelikeofapatientshouldbeconsidered．

［0100］Furtherm0e，theantibodyorthefunctionalfrag－ mentofthepresentlnVentionmaybemixedintotoothpaste OranintraoralwashandthenappliedtoareglOnaffectedby Periodontaldiseases．Theantibodyorthefragmentthereof Can also be appliedintheformOfafunctionalfbodby mixlngtheantibodyorthefragmentintofbod，drink，Orthe like．

［0101］FurthermOre，theantibodyorthefunctionalfrag－ mentthereofofthepresentinventioncanalsobeusedasa diagnostic agentfor periodontaldiseases．For example， WhetherornotP ginglValisis presentintheperiodontal POCketcanbedetectedusingtheantibodyorthefunctional fragmentthereofofthepresentinvention．Suchdetection CanbecarriedoutbycollectlngPlaqueintheperiodontal POCketandthendetectlngthepresenceofPginglValisinthe PlaquebyaknowninlmunOaSSaySuChasEIA，RIA，Oran immunOagglutinationmethod．

［0102］HereaRer，the presentinventionis describedin detai1byreferringtoexamPles，butisnotlimitedtoembodi－ mentsdescribedintheexamPles．

EXAMPLEI

PreparationofRecombinant40－kDaOMP（r40－kDa OMP）

［0103］Recombinant40－kDa OMP（r40－kDaOMP）was PrePared as fo1lows．Escherichia coli（K－12）having a recombinantplasmidpMD125preparedbyincorporationof fu11－1engthr40－kDaOMPDNA（DNADataBankofJapan： accessionNo．ABO59658）intoavectorwasculturedinan LBmedium（1％tryptone（producedbyBecton，Dickinson andCompany），0．5％yeast extract（producedby Becton， DickinsonandCompany），andO・5％NaCl）containirtglO LJmLtetracycline．Bacterialbodieswerecollecteduslnga CentrifugeandthendisruPtedbyultrasonication．Asuper－ natantwhereinthebacterialbodieshadbeendisruPtedwas Obtaineduslng a Centrifuge andthenr40－kDa OMPwas PurifiedaccordingtothemethodofKawamOtOetal．，（Int．J． Biochem．1991Ⅵ）123：1053）．Thethuspreparedr40－kDa OMPwas subjectedto substitutionwith PBS（－）using a dialysismembrane（molecularweightoflO，0000rlessasa Cut－0住producedbySpectrumLaboratoriesInc．）．Thus，a Purifiedproteinofaslnglebandcorrespondingtoamolecu－ 1arweightof40，000wasobtainedbySDS／fAGEelectro－ phoresis．

EXAMPLE2

ProductionofHuman－antibody－PrOducingMice

［0104］Mice usedforinlmunizationhave genetic back－ groundsuchthattheyarehomozygousfbrbothdisruPtlOnin theendogenousIgheavychainanddisruPtlOninthel｛1ight Chain．FurthermOre，the mice simultaneously retained a Chromosome14fragment（SC20）containing ahumanIg heavychainlocusandahumanIgl｛Chaintransgene（KCo5）． Thesemicewereproducedby crosslngmice ofalineA havingahumanIgheavychainlocuswithmiceofalineB havingahumanIgl｛Chaintransgene．ThemiceofthelineA arehomozygousforbothdisruPtlOnintheendogenousIg heavychainanddisruPtioninthel｛1ightchain．Furthermore， themice ofthelineAretainachromosome14fragment （SC20）thatcanbetransferredtoprogenyandisdescribed inthereportofTomizukaetal．（Tomizuka．etal．，Proc．Nati． Acad．Sci．U．S．A．，2000Ⅵ）197：722），forexamPle．Further－ more，the mice oftheline B are homozygous fbr both disruPtlOnintheendogenousIgheavychainanddisruPtlOn inthel｛1ightchain．ThemiceofthelineBretainahuman IgKchaintransgene（KCo5）andaredescribedinthereport OfFishwildetal．，（Nat．Bioteclm01．，1996Ⅵ）114：845），hr example．Mouseindividuals（Ishida＆Lonberg，IBC’sllth AntibodyEngineering，Abstract2000）forwhichthehuman IgheavychainandtheKlightchainarpsimultan？OuSly detectedinserumWereObtainedbycrosslngmalemlCeOf thelineAwithfemalemiceofthelineBorcrosslngfemale miceofthelineAwithmalemiceofthelineB．Suchmice WereuSedinthefo1lowlnglnlmunOloglCalexperiments．In addition，theabovehuman－antibody－PrOducingmicecanbe acquiredfromKirinBreweryCo．，Ltd．basedonconclusion ofa contract．

EXAMPLE3

PreparationofaHumanMonoclonalAntibody Against40－kDaOMP

［0105］Monoclonalantibodiesinthisexamplewerepre－ Paredaccordingtogeneralmethodsdescribedin”Introduc－

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tion to MonoclonalAntibody ExperimentalProtocoIs （MonoclonalKo－taiJikken So－Sa叫ノu－mOn）（written by

ThmieAndo，issuedbyKODANSHA，1991）andthelike． r40－kDa OMP preparedin Examplelwas used as an immunOgen．As animals to beimmunized，the human－ antibody－PrOducingmice（producedinExample2）produc－ 1nghumaninlmunOglobulinwereused．

［0106］r40－kDaOMPpreparedinExamplelwasmixed WithanRIBIaqjuvant（producedbyCorixaCorporation）． Thehuman－antibody－PrOducingmiceweresubjectedtoini－ tialinlmunizationbyintraperitonealadministrationof20トLg Ofr40－kDaOMP．Boosterinlmunizationwascarriedoutby 4timesintraperitonealadministrationofthemixedsolution Ofr40－kDaOMPandtheRIBIaqjuvanteverylto2weeks aRerinitialinlmunization．FurthermOre，3 days befbre Obtainlngthespleencellsasdescribedbelow，boosterinlmu－ nization was carried out by tai1intravenouslI勺eCtion of r40－kDa OMP．

［0107］Spleensweresurgicallyobtainedfromtheinlmu－ nizedmice．Thethuscollectedspleencellsweremixedwith mousemyelomaSP2／0（ArCCNo．：CRL1581）ataratioof 5：1．Thus，Cellfusionwas carriedoutuslngPOlyethylene glycol1500（produced by Boehringer Mannheim）as a fusionagent，therebypreparlngmanyhybridomas．Hybri－ domaswereselectedbyculturlngtheminaHAr－COntainlng DMEMmedium（producedbyGibcoBRL）containinglO％ fetalcalfserum（FCS），hypoxanthine（H），aminopterin（A）， andthymidine（T）・FurthermOre，Singlecloneswere

．

DMEM medium．Culture was carried outin a 96－Well

microtiterplate（producedbyBecton，DickinsonandCom－ Pany）．Selection（screening）ofhybridomaclonesproducing anti－r40－kDaOMPhumanmOnOClonalantibodiesandchar－ acterizationofahumanmOnOClonalantibodyproducedby eachhybridomawerecarriedoutbymeasurementsuchas enzymelinkedimmun0Otbentassay（ELISA）orfluores－ CenCeaCtivatedcellsortlng（FACS）describedlater．

［0108］Human－mOnOClonal－antibody－PrOducing hybrido－ maswerescreenedforbyELISA，SPeCificallybyenzyme linkedimmun0Otbentassay（ELISA）andfluorescenceacti－ Vated cellsortlng（FACS）described below．Hybridomas PrOducinghumanmonoclonalantibodiesthathaveahuman immunOglobulinYChain（hIgY），ahumaninlmunOglobulin lightchainl｛，andspecificreactivltytOr40－kDaOMPwere Obtained．Inaddition，1n any Ofthefo1lowlng ExamPles， includingthis Example，andintables orfigures glVentO ShowtestresultsintheExamPles，hybridomaclonespro－ ducingeachhumananti－40－kDaOMPmonoclonalantibody OfthepresentinventionweredenoteduslngSymboIs．The hllowinghybridomaclonesrepresentslngleclones：h13－17， 5－89－2，a44－1，andl－85－16．Oftheseclones，3hybridoma Clones，h13－17，5－89－2，and a44－1，Wereinternationally depositedattheInternationalPatentOrganismDeposltaryOf the NationalInstitute ofAdvancedIndustrialScience and

Tbclm0logy（Tsukuba Centra16，1－1－1Higashi，Tsukuba， Ibaraki，Japan）underthe BudapestTreaty．The accession nos．ofthehybridomaclonesh13－17，5－89－2，anda44－1are FERM BP－8325，FERM BP－8323，and BP－8324，reSPeC－ tively（asofMar．11，2003）．

EXAMPLE4

Detection of a Monoclonal Antibody having a HumanhlmunOglobulinYChain

［0109］50トLlofr40－kDaOMP（1トLg／m150mMNa2HCO3） PreParedinExamplelwasaddedtoeachwellofa96－Well microplatefbrELISA（Maxisorp，PrOducedbyNunc），hl－ lowedby30minutes ofincubationatroomtemperature． r40－kDa OMP was thus adsotbed onto the microplate． Subsequently，thesupernatantSWerediscarded．Ablocking reagent（SuperBlockTMBlockingBu正己rproducedbyPierce Bioteclm0logyInc．，）wasaddedtoeachwell，hllowedbylO minutesofincubationatroomtemperature．Thus，Sitesto which no r40－kDa OMP had bound were blocked．In this

manner，amicroplatewaspreparedsothateachwellwas COatedwithr40－kDaOMP．Furthermore，PginglValis381 strain was anaerobically cultured in Tripticase soy broth G3rOduced by BBL）supplemented with5トLg／mL hemin G3rOducedbySigma），0．5トLg／mLvitaminK，andO．5％yeast extract（producedbyDiftoLaboratories）at370C．Pgin－ glValiswasgrowntothemid－logphase．Subsequently，the bacterialcellswerecollectedbycentrifugation（10，000×g，

10minutes，and40C．），hllowedbyheattreatmentat600C． for30minutes．Next，thecellswerere－SuSPendedinPBS andthensubjectedtosonicationonicefbr15minutesuslng an ultrasonic homogenizer（Branson Sonifier250）．The ultrasonicated resultant was centrifuged（100，000×g，30

minutes，and40C．）andthenthesupernatantwasfi1tered （0．22トLm）．Theconcentrationoftheultrasonicatedresultant WaSfoundbymeasurementofabsotbanceat280nmand thencalculationwherelmg／mlwas determinedtobean OPtimaldensity（0．D．）ofl．0．UltrasonicatedP gingivalis （50トLg／m150mMNa2HCO3and50トLl／well）wasaddedto eachwellofa96－WellmicroplateforELISA（Maxisorp， PrOducedbyNunc），hllowedby30minutesofincubationat roomtemperature．Thus，theultrasonicatedPginglValiswas adsotbedtothemicroplate．Subsequently，SuPernatantSWere discarded．A blocking reagent（SuperBlockTM Blocking Bu正己rproducedbyPierceBioteclm0logyInc．，）wasadded toeachwell，fo1lowedbylOminutesofincubationatroom temperature．Each wellwas washed twice with O．1％ Tween20－COntainingphosphatebuffer（PBS－T）．50トLlofthe

CulturesupernatantOfeachhybridomawasaddedtoeach Wellofthemicroplatecoatedwithr40－kDaOMPorultra－ SOnicatedPginglValis，丘1lowedby30minutesofreaction atroomtemperature．Eachwellwasthenwashedtwicewith PBS－T．Agoatanti－humanIgGF（abT）2antibody（produced by BiosourceInternational）1abeled with peroxidase was diluted2000－fo1dbyuslngPBS－TcontainlnglO％Block Ace（producedbyDainipponPharmaCeuticalCo．，Ltd．）．50 L1lofthesolutionwasaddedtoeachwell，fo1lowedby30 minutesofincubationatroomtemperature．Themicroplate WaSWaShedthreetimeswithPBS－T．100トLlofacolonng Substratesolution（TMBproducedbyDAKOCorporation） WaSaddedtoeachwell，fo1lowedby20minutesofincuba－ tionatroomtemperature．50トLlof2Msulfuricacidwas addedtoeachwellsoastostopthereaction．Absorbanceat awavelengthof450nm（refbrencewavelength：570nm） WaS meaSuredusing a microplate reader（MTP－300pro－ ducedbyCoronaElectricCo．，Ltd）．Asaresult，3000rmOre anti－r40－kDaOMPantibodyclonescouldbeobtained．Some ofthe clones are shownin FIG．1．

US2007／0036734Al Feb．15，2007

Anti－r40－kDa OMP antibody－PrOducing hybridomas were adapted to an eRDF medium（produced by KYOKUTO PHARMACEUTICALINDUSTRIALCO．，uD．）contain－ ingcattleinsulin（5トLg／ml，PrOducedbyGibcoBRL），human transfbrrin（5トLg／ml，PrOducedby Gibco BRL），ethanola－ mine（0二PlmM，PrOducedbySigma），andsodiumselenite （2・5×10mM，PrOducedbySigma）・ThehybridonチaSWere Culturedinasplnnerflask．WhentheviablecellratlOOfthe hybridomas reached90％，a Culture supernatantwas col－ 1ected．ThecollectedsupernatantWaSfi1teredusingalOトLm fi1terandaO．2トunfi1ter（producedbyGelmanScience，Inc．）， therebyremovlngmiscellaneoussubstancesincludinghybri－ domasandthelike．

［0113］Theanti－r40－kDaOMPantibodieswヲrePurified fromtheaboveculturesupernatantbythefo1lowlngmethod． TheculturesupernatantCOntainlngtheanti－r40－kDaOMP antibodiesweresubjectedtoa伍nitypurificationuslngthe HyperDProteinAcolumn（producedbyNGKINSULA－ TORS，uD・），PBS（－）asanadsorptionbuffer？CCOrdingto theattachedinstruCtions，andO．1M sodiumCltratebu正己r G3H3．5）asanelutionbuffer．Thethuselutedfractionwas aqjustedtoaroundpH7．2bytheadditionoflMTris－HCI G3H8．0）．Thethuspreparedantibodysolutionwassubjected to substitution with PBS（－）using a dialysis membrane （molecular weight oflOOOO as a cut－0住produced by SpectrumLaboratories，Inc．，）andthenfi1ter－Steri1izedusing a MILLEX－GV membranefi1ter（produced by Millipore Corporation）withapore size ofO．22トLm．Thus，Purified anti－r40－kDa OMP antibodies were obtained．The concen－

trationofthepurifiedantibodieswasfbundbymeasurement Ofabsorbanceat280nmandcalculationwherelmg／mlwas determinedto bel．450．D．

EXAMPLE

PreparationofIsotypeControlAntibodies

［0114］Inamannerヲimilartotheabovemethod，human－ antibody－PrOducingmlCeWereinlmunizedwithDNP－KLH andthenthespleencellsofthemicewerefusedwithmouse myeloma SP2／O cells．Thus，many hybridomas werepre－ Pared．Specifically，anti－humanIgGl，IgG2，andIgG4anti－ bodieswereprepared．

EXAMPLE9

InhibitionofCo－aggregationofPginglValisby anti－r40－kDaOMPAntibody

［0115］Atestofinhibitingthecoaggre甲tionofPgingi－ ValiswasimplementedbyamodifiedverslOnOfthemethod OfEllenR．Retal．（Infbct．hlmun．1989；57：1618－1620）． Pgingivalis381（（1）Postalcode：271－8587，2－870－1，Nishi， Sakae－maChi，Matsudo－Shi，Chiba，Japan，Prof二Nobumitsu Abiko，Department ofBiochemistry and MolecularDen－ tistry，NIHONUNIVERSITYSchoolofDentistryatMat－ Sudoor（2）Postalcode：951－8514，5274，2－ban－Cho，Gakko－ machi－do－ri，Niigata－Shi，aCquiredfrom Prof二 Hiromasa Ⅵ）Shie，DepartmentofOralBiologlCalScience，Divisionof Periodontology，Niigata University Graduate Schoolof Medicaland DentalSciences）vesicles（0．7ng／mL）were PreParedaccordingtothemethodofHiratsukaetal．（Arch． Oral．Biol．1982，37：717－724），fo1lowedby30minutesof reactionwitheachantibodyat370C．FurthermOre，Actino－ nv）CeS Viscosus＠．viscosus，ArCC19246）was anaerobi－

EXAMPLE5

DetectionofaMonoclonalAntibodyhaving HumanhlmunOglobulinLightChainl｛（IgLl｛）

［0110］50トLlofr40－kDaOMP（1トLg／m150mMNa2HCO3） PreParedinExamPlelwasaddedtoeachwellofa96－Well microplatefbrELISA（Maxisorp，PrOducedbyNunc），hl－ lowedby30minutes ofincubationatroomtemperature． r40－kDa OMP was thus adsorbed onto the microplate． Subsequently，thesupernatantSWerediscarded．Ablocking reagent（SuperBlockTMBlockingBu正己rproducedbyPierce Bioteclm0logyInc．）wasaddedtoeachwell，hllowedbylO minutesofincubationatroomtemperature．Eachwellwas WaShedtwicewithPBS－T．50トLloftheculturesupernatant Ofeachhybridomawasaddedtoeachwellofthemicroplate COated with r40－kDa OMIlfo1lowed by30minutes of reaction．EachwellwasthenwashedtwicewithPBS－T．50

L1lofagoatanti－humanIgl｛antibody（diluted2，000－fo1dand

PrOducedbyBiosourceInternational）1abeledwithperoxi－ dasewas addedto eachwell，fo1lowedby30minutes of incubationatroomtemperature．ARerwashingthreetimes WithPBS－T，100トLlofasubstratebuffer（TMBproducedby DAKOCorporation）wasaddedtoeachwell，fo1lowedby20 minutesofincubationatroomtemperature．50トLlof2M sulfuric acid was added to each well so as to stop the reaction．Absotbanceatawavelengthof450nm（reference WaVelength：570nm）was measured using a microplate reader（MTP－300producedbyCoronaElectricCo．，Ltd）．

EXAMPLE6

Identificationofthe Subclass ofeachMonoclonal Antibody

［0111］50トLlofr40－kDaOMP（1トLg／m150mMNa2HCO3） PreParedinExamPlelwasaddedtoeachwellofa96－Well microplatefbrELISA（Maxisorp，PrOducedbyNunc），hl－ lowedby30minutes ofincubationatroomtemperature． r40－kDa OMP was thus adsorbed onto the microplate． Subsequently，thesupernatantSWerediscarded．Ablocking reagent（SuperBlockTMBlockingBu正己rproducedbyPierce Bioteclm0logyInc．）wasaddedtoeachwell，hllowedbylO minutesofincubationatroomtemperature．Eachwellwas WaShedtwicewithPBS－T．50トLloftheculturesupernatant Ofeachhybridomawasaddedtoeachwellofthemicroplate COated with r40－kDa OMIlfo1lowed by30minutes of reaction．Each wellwas then washed twice with PBS－T．

Subsequently，50トLlofasheepanti－humanIgGlantibody，a Sheepanti－humanIgG2antibody，aSheepanti－humanIgG3 antibody，Or a Sheep anti－humanIgG4antibody（diluted 2，000－fo1d and produced by The Binding Site Limited） 1abeledwithperoxidasewasaddedtoeachwell，fo1lowedby 30minutesofincubationatroomtemperature．Afterwash－ 1ngthreetimes withPBS－T，100トLlofa substratebu正己r （TMBproducedbyDAKOCorporation）wasaddedtoeach Well，fo1lowedby20minutes ofincubationatroomtem－ Perature．50トLlof2Msulfuricacidwasthenaddedtoeach Wellsoastostopthereaction．Absotbanceatawavelength Of450nm（referencewavelength：570nm）wasmeasured usingamicroplatereader（MTP－300producedbyCorona ElectricCo．，Ltd）．

EXAMPLE7

PreparationofeachAntibody

［0112］Culture supernatantS COntaining the anti－r40－kDa

OMPantibodieswerepreparedbythefbllowlngmethod．

US2007／0036734Al Feb．15，2007

10

Callyculturedin37mg／mLbrainheartinfusion（produced byBBL）supplementedwith5mg／mLyeastextract（pro－ ducedbyBBL）at370C．A．viscosuswaspreparedtohave anabsorbanceofl．5（wavelengthof500nm）usingPBS． Thethusprepared50トLLofA．viscosuswassuspendedinan equlValent amount OfPBS．50トLL ofP glnglValis381 VeSiclesthathadreactedwitheachantibodywereaddedto the solution，hllowed bylO minutes ofreaction on a flocculation slide at370C．ARer reaction，aggregation activltyWaSeValuatedviathenakedeyeoralightmicro－ SCOPe．Inhibition activitycriteria（score：O to4）werein accordancewiththemethodofCisar．J．0．etal．，（Infbct． hlmun．，1979Ⅵ）133：467）．As aresult，13anti－r40－kDa OMPantibodiesshowedactivltyOfinhibitingthecoaggre－ gationofPginglValis381vesicleswithA．viscosus．From among antibodies having such activity，h13－17，5－89－2， a44－1，andl－85－16havingstrongcoaggregation－inhibiting activitywereselected（Thblel）．Thefbllowingexperiment WaSCOnductedusingthese4antibodies．

TABLEl

1077andll19（produced by Sigma）．Furthermore，the remaining erythrocytes were hypotonically hemolysed in ice－COOledredbloodcelllysissolution（10mMTris，10mM KCl，1mMMgC12，andpH7・4）・ARerrecoveryofosmotic PreSSure With PBS，the resultant was washed and then preparedtoaconcentrationof2×106／mLofhumanneutrO－

Phils．Thethuspreparedneutrophilswereinlmediatelysub－」eCtedtothenextphagocytosistest．

［0117］Theanti－r40－kDaOMPantibodyoracontrolanti－ body（1Llg／mLand5LIL）was addedto2×107cfu／mL FITC－1abeledPginglValis381，fo1lowedby30minutesof reaction at370C．ARer washing twice with PBS，the resultantwasre－SuSPended．HumanneutrophilsandFITC－ 1abeledPginglValisweremixedataratioofl：20andthen Culturedfor30minutesat40C．or370C．ARerphagocytlC reaction，10，000neutrophilswereincorporatedbyFACScan G3rOducedbyBecton，DickinsonandCompany）andthenthe fluorescenceintensityofFITCwasmeasured，SOthatphago－ Cyticabilitywasevaluated．Thepercentageofneutrophils G3hagocytosisrate）thathadconductedphag？CytOSiswas foundbythe fbllowing equation．PhagocytosIS rate（％）＝ G3erCentage（％）ofFITC－POSitiveneutrophils at370C．）－

G3erCentage（％）ofFITC－POSitiveneutrophilsat40C．）．Asa result，itwasobserved（Thble2）thatwhencomparedwith thecontrolantibody，allantibodieshadaneffectofenhanc－ ing

．

reportedinSitoS．etal．，Gen．Pharmacol，1997Ⅵ）128：675） WaSCOmParedwiththatofhuman40－kDaOMPantibodies （h13－17，5－89－2，and a44－1）using the same evaluation

SyStem．As a result，the mouse antibody Pg－OmPA3was foundtohaveveryweakactivityofenhancingthephago－ Cytic abilityofhumanneutrophils．Thus，thehumananti－ bodieswereshowntobesuperiortothismouseantibody （FIG．2）．

nBLE2

Eachanti－40kDa－OMPantibody’sactivityof

inhibitingthecoaggregationofPgtnglValis

Tablel

Activityofinl1ibiting

COaggregation Subclass Clone

IgGl（K）

IgGl（K）

IgGl（K）

IgGl（K）

a44－1

血13－17

1－85－16

5－89－2

Antibodyconcentration：a44－1（193Llg／mL），h13－17（175Llg／mL），1－86－16

（161ト釘mL），5－89－2 ．

gation，thestrongertheactivity

EXAMPLElO

ActivationofPromotlngHumanNeutrophilic PhagocytosisbyAnti－r40－kDaOMPAntibody

［0116］Phagocytosistestswereimplementedbyamodi－ fiedversionofthemethodofPerticarariS．etal．，（Cytometry 1991；12：687－693）．ThePgingivalis381strainwasanaero－ bicallyculturedinTripticasesoybroth（producedbyBBL） SuPPlementedwith5トLg／mLhemin，0．5トLg／mLvitaminK， andO．5％yeastextract（producedbyDiftoLaboratories）at 370C．P glnglValis was grown to the mid－log phase． Subsequently，thebacterialcellswerecollectedbycentrifu－ gation（10，000×g，10minutes，40C．），hllowed by heat

treatmentat600C．hr30minutes．ARerwashingtwicewith PBS，thecellswereaqjustedtoaconcentrationof2×108 Cfu／mL．1mLofFITC（producedbyMolecularProbesInc．，） PreParedtoaconcentrationoflmg／mLwithaO．1Msodium Carbonatebuffer（pH9．6）wasaddedtolmLofasolution COntainlngaSuSPenSionofPginglValis381，fo1lowedby30 minutesofcultureat370C．Afterwashingthreetimeswith PBS，FITC－1abeled P ginglValis381was prepared to a concentrationof2×108cfu／mL・Bindingoftheanti－r40－kDa OMPantibodytotheFITC－1abeledPginglValis381wasat the samelevelas thatfbrunlabeledP ginglValis381．A methodfbrseparatinghumanneutrophilswascarriedoutby COllectlnghumanperipheralvenousbloodusingaheparin－ COatedvacuumtubefbrbloodcollectionandthencarrylng Outdouble－densitygradientcentrifugationusingHistopaque

Eachanti－40kDa－OMPantibody’sactivityofpromotingphagocytosis

OfPginglValisbyhumanPOlymorphonuclearneutrophils

nble2

Clone PMNphagocytosisrate

76．2％＊

83．0％＊

78．6％＊

81．3％＊

49．8％

5－89－2

1－85－16

a44－1

血13－17

ControlIgGl

＊ThePMN（Polymorphonuclearneutrophils）phagocytosisratewassig－

nificantlyhigherthanthatinthecaseofthecontrol．

EXAMPLEll

Comparisonbetweenr40－kDaOMPAntibodyand Patient’sSeruminTermsofActivityofBindingto

Pg∫〝gJVdJ∫∫

［0118］Fourr40－kDaOMPantibodiesweIアCOIモParedwith theseraofchronicperiodontaldiseasepatlentSlntermSOf theintensltyOfbindingtoPginglValisbyELISA，thesame methodemployedinExample4．50トLlofeachantibody（150 Llg／mL）orIgG antibody（Kobayashi，T．et al．，Infbct． Immun．，2001Ⅵ）169：2935）derivedfromthepatients’sera thathadbeenapproprlatelydilutedwasaddedtoeachwell Ofamicroplatecoatedwithr40－kDaOMPorultrasonicated

US2007／0036734Al Feb．15，2007 11

PginglValissothatreactionbetweenthemcouldtakeplace． Afterwashing，boundantibodiesweredetecteduslngPer－ oxidase－1abeledgoatanti－humanIgGF（abT）2antibody（pro－ ducedbyhlmun0－BiologicalLaboratoriesCo．，Ltd．）anda COloring substrate solution（TMB）．As a result，it was revealedthat，Whenconcentrations atwhichthepatients’ SerahadboundtoadegreeequlValenttothatinthecaseof r40－kDa OMPwerecomparedwiththe concentrations of r40－kDaOMPsthathadboundtoPginglValis，allantibodies hadmoreintensiveactivityofbindingtoPgingivalisthan thatofpatients’sera（FIG．3）．

EXAMPLE12

Cross－ReactivltyOfeachAnti－r40－kDaOMP AntibodytoHumanBloodCells

［0119］Thecross－reaCtivityofeachmonoclonalantibody tohumanbloodcellswasexaminedbyFACSanalysIS．10 mLofhumanperipheralbloodcontainlnglmLofheparin （producedbyNovoA／S）wasdiluted2－hldwithlOmLof PBS（－）・The solutionTaS SuPerPOSed on20mLofa Ficoll－PaquePLUSsolutlOn（producedbyAmershamPhar－ maciaBiotech）．After30minutesofcentrifugationat1500 r．p．m．，amOnOnuClearleukocytefractionwascollectedand thenwashedtwice withPBS（－）．Thethus prepared cells weresuspendedataconcentrationof2×106／mlinaPBS

StainingBuffer（SB）containingl％ratserum，0．1％NaN3， and2％FCS．Thecellsuspension（100トLl／well）was dis－ PenSedintoa96－Wellround－bottomedplate（producedby Becton，DickinsonandComp㌍y）・Theh13－17，5－89－2， a44－1，Orl－85－16antibodywaslnCubatedataconcentration Of5トLg／mLaticetemperaturefor30minutes．ARerwashing twicewithSB，theresultantwassuspendedin300トLlofa FACSbuffer．ThereactivltyOfeachantibodywasexamined by FACS（FACScanproducedby Becton，Dickinsonand Company）．Asaresult，nOantibodieswerefbundtobindto humanperipheralbloodcells．Basedontheseresults，1tWaS inferredthatallantibodiesmerelycausesidee正己ctsupon administrationto humans．

EXAMPLE13

BindingActivltyOfAnti－r40－kDaOMPAntibodyto Pg∫〝gJVdJ∫∫

［0120］Binding of each antibody to P gingivalis was evaluatedusingasurfhceplasmionresonapcemethod（BIA－ CORE，PrOducedbyBIACOREInternatlOnalAB）．Sensor Chips（CM5）weresubjectedtoinlmObilizationaccordingto experimental protocols by an amine coupling method （ImmobilizedamOuntS：CIgGl＝9097RU，a44－1＝7355RU， h13－17＝7473RU，5－89－2＝7595RU，andl－85－16＝7870RU）． PginglValiswastreatedat600C．hr30minutessoastodie andthenthebacterialbodieswerepreparedtoaconcentra－ tionofO．D．550nm＝0．4withalOmMTris－HCl／150mM

NaClbu正己r（pH8．0）．Theresultantsweresuppliedataflow rateoflOトLL／min．Asaresult，thedissociationrateinthe CaSeOf5－89－2wasslowerthanthoseoftheotherantibodies， Showing that the5－89－2antibody strongly binds to P gingivalis（FIG．4）．Inviewofsaliva’sactiontoeliminate antibodiesthatwillbeexertedupontreatmentofperiodontal diseases，aPrefbrableantibodyisthe5－89－2antibodyshow－ 1ng Slow dissociation，hr examPle．Therefbre，it was inferredthatthe5－89－2antibodyshowsahighlytherapeutic e正己ctwhenitis administeredto ahuman．

EXAMPLE14

Anti－r40－kDaOMPAntibodies’Activityof InhibitingtheBindingofHemintoP

［0121］Whether or not anti－r40－kDa OMP antibodies inhibitthebindingofheminto40－kDaOMPwasevaluated usingasurfhceplasmonreson竺nCemethod（BIACORE， PrOducedbyBIACOREInternatlOnalAB）．r40－kDaOMP WaSinlmObilizedbyanaminecouplingmethod（RU＝3500）． FIG．5showstheresults ofsupplyingeachantibodypre－ Paredat20トLg／mLwithalOmMTris－HCl／150mMNaCl （T－B，PH8．0）bu正己rataflowrateof20トLL／mandthen SuPPlying5トLg／mLhemin（producedby Sigma）prepared WithaTJBbu正己r．FIG．5showsasensorgramWherethetime Whenthesupplyofheminwasinitiatedwasdeterminedto beOseconds．Whenh13－17hadbeenpreviouslyboundto r40－kDa OMIIsignificantlyless binding of hemin was Observed comparedwiththe case ofcIgG．Onthe other hand，inthecaseoftheother3clones，bindingofr40－kDa OMPtoheminwasobservedtothesamedegreeasthatin thecaseofacontrol，eVenWhentheyhadbeenpreviously boundtor40－kDaOMP．Theseresultssuggestedthath13－17 inhibitsthebindingofhemintor40－kDaOMP．Furthermore， FIG．6showstheresultsofexaminlngWhetherornoth13－17 inhibitsthebindingofhemintor40－kDaOMPwhenh13－17 andheminaresimultaneouslycausedtoreact．h13－17and heminwerepreparedtoconcentrationsof20トLg／mLand5 Llg／mL，reSPeCtively，WithaT－Bbu正己r．Theyweresupplied tor40－kDaOMP－inlmObilizedsensorchipsataflowrateof 20トLL／m．a44－1antibodyhavingadissociationratethatis almostthesame（seeFIG・4）asthatofh13－17waヲuSedas acontrolantibody．Asaresult，Whensignalsatatlme（380 SeCOnds）nearthetimeoftheequilibriu竺aRerdi仇1Sionof analytes were observed，the binding slgnalofhemin to r40－kDaOMPwas255RUinthepresenceofh13－17，Which WaSalmostthesameasthat（252RU）inthecaseofh13－17 alone．Thedifferenceinthesignals（255RU－252RU＝3RU） WaSClearlylowerthanthe signal（30RU）inthecaseof COntrOIIgGl＋hemin at this time，SuggeStlng alack of binding ofhemin to r40－kDa OMP．On the other hand， regardingthesignalinthecaseofthecontrola44－1at380 SeC．，thesumofthesignalinthecaseofa44－1andthesignal inthecaseofcontroIIgGl＋heminwas180RU（148RU＋32 RU）．Thesignalinthecaseofa44－1＋heminwas170RU， Whichwasclosetothetheoreticalvalue（180RU）atthetime When no antibodies were competlng With hemin．These resultssuggestedthattheh13－17antibodystronglyinhibits thebindingofhemintoOMP40．

EXAMPLE15

SuppressiveEffectofAnti－r40－kDaOMP AntibodiesonRatExperimentalPeriodontitis

［0122］RatnヲutrOPhilswereseparatedfr0ⅠチratPeripheral bloodbydensltygradientcentrifugationuslngaLympho－ 1ite－Rat（produced by Sigma）・In a

．

Phagocytosis of each anti－r40－kDa OMP antibody was evaluated．Thustheactivitywasobserved．Hence，aneXPeri－ mentalsystemfbrperiodontitiswaspreparedbyinoculatlng ratoralcavitieswithPginglValistoinducealveolarbone resorptlOn．Throughtheuseofsuchsystem，theeffectofthe anti－r40－kDaOMPantibodies（havingstrongabilitytobind

US2007／0036734Al Feb．15，2007 12

to，aCtivltyOfinhibitingcoaggregationabilityof，andactiv－ ityofpromotingneutrophilicphagocytosisofPgingivalis） tosuppressratexperimentalperiodontitiswasexaminedin invitroexperiments．3－Week－01dSprague－DawleySPFrats （6ratspefgrOuP）wereusedforinductionandinfectionof rat expenmentalperiodontitis due toinoculation withP glnglValis．After observation of health conditions，Sul－ famethoxazolewithafinalconcentrationoflmg／mlwas mixedwith200トLg／mltrimethoprlminionexchangedwater． Themixedsolutionwasadministeredasdrinkingwaterfor lweek，thereby reducing normalbacteria ofthe mouth． Subsequently，antibiotic－freeion exchanged water was administeredfbr3days．Abactereialsolution（109cFU／ml） OfPgingivalisstrain（ArCC33277）waspreparedwitha5％ Carboxymethylcellulose（CMC）solutlOnPreParedwith PBS．ThebacterialsolutionwasdirectlyadminlSteredinto ratoralcavities3timesevery2days．Agroup（sham）that hadnotbeeninoculatedwithPginglValiswasadministered With only5％CMC solution and raisedunderthe same COndtions．Antibodieswereadministeredasfo1lows．a44－1， h13－17，and5－89－2antibodies were each prepared to a COnCentration ofO．5mg／ml．3antibodies were mixedin equalamounts，theresultantwasdilutedwitha5％CMC SOlution，andthenthesolutionwasadministeredtoagroup． ADNPhumanantibody（IgGl）preparedtobeaconcentra－ tionofO．5mg／mlwitha5％CMCsolutionpreparedusing PBSwasadministeredtoacontrolgroup．Thesegroupswere used．Administrationtookplace9timesintotal；SPeCifically， administrationwasperhrmedeverydayfrom2daysbefbre thestartofadministrationofPginglValisto2daysafterthe finaladministration．Inaddition，atthetimeofinoculation uslng P ginglValis，the antibodies were administeredlO minutesafterinoculationwiththebacteria．Allratswerefbd

ad libitunm diets and drinking water and raised under COnditionsof12－hourlightanddarkcyclesatatemperature Of230C．andhumidityof60％．

［0123］ThepresenceofPgingivalisin ．

eachratoralcavltyWaSWipedwithaswabfor30seconds to collect plaque bacteria．DNA was extracted uslng ISOPLANT（producedbyNIPPONGENECO．，uD．）．The extractedDNAwasdissoIvedin20トLLofaTl。EISOlution andthenstored．Primerswerepreparedbasedonthenucle－ Otide sequence of16S rRNAaccording to the report of Ashimotoetal．，（Ashimoto，A．etal．，OralMicrobiolhlmu－ nol．，1996Ⅵ）111：266）．PCRreactionwasperhrmed，after heatingat950C．for5minutesfbrdenaturation，in35cycles， eachofwhichconistedof950C．for30seconds，600C．for lminute，and720C．hrlminute．

［0124］Boneresorption㌣aSmeaSuredasfbllows・Onday 42aRerthefinalinoculatolnWithPginglValis，allratswere SaCrifiedbydecapltationandbloodlettlngunderetheranes－ thesia．TheamountsofalveolarboneresorptlOnWereeValu－ atedbymeasuringdistances（sevenpositions）betweenthe Cement－enameljunctionofamaxillarymolartoothportion andthecrestofthealveolarridge．Thecranialboneswere heatedunderanatmosphericpressureof2fbrlOminutes andinlmerSedin a3％sodiumhypochlorite solution to remove soR tissues．The alveolar bones were stained and driedwithal％methylenebluesolution．Thethusprepared SamPles were measuredunder a digitalHD microscope （stereoscopicmicroscopeproducedbyKEYENCECORPO－ RArION）atx40magnification．Valuesmeasuredat7posi－ tionswereaveragedtoobtaintheboneresorptlOnamOunt

Perratindividual．Themeanvalueobtainedbyaveraglngthe meanvaluesof6ratswasdeterminedtorepresentthebone resorptlOnamOuntOfanexperimentalgroup anddenoted withmilimeters．Measurementwas carried out three times

forthesameSamPleandthenthemeanvalueandstandard error（SE）werefbund・Statisticanalysiswascarriedout uslngFisher’sPLSD（StatView）．

［0125］Asaresult（FIG．7），aClearlysignificantincrease WaSObservedinboneresorptionamOunt（p＜0．01）inthecase Ofagroup towhichP ginglValishadbeenadministered， COmParedwithagrouptowhichnonehadbeenadminis－ tered．FurthermOre，SlgnificantlysuppressedboneresorptlOn WaS COnfirmedbythe administration ofthe anti－r40－kDa OMPantibodies．Specifically，theamountofboneresorptlOn inthe case ofa group fbrwhichthe anti－r40－kDa OMP antibodieshadbeenusedataconcentrationofO．5mg／ml WaSalmostthesameasthatinthecaseofagrouptowhich noPginglValishadbeenadministeredandinwhichalmost noboneresorptlOnduetoadministrationofPginglValiswas Observed．In the case of a group to which the control antibody（DNPantibody）hadbeenadministered，theamOunt OfboneresorptlOnWaSSOmeWhatdecreasedcomparedwith agrouptowhichonlyPginglValishadbeenadministered． However，nO Slgnificantdi正己rencewas observedbetween thetwo．Basedontheaboveresults，1tWaSreVealedthatin this experimentuslngboneresorptonas anindex，mOnO－ ClonalantibodiesagalnSttheOMP40antlgenCanSuPPreSS ratalveolarboneresorptlOn．FurthermOre，Cleardecreases WereObservedintheamountSOfboneresorptlOnWiththe administrationoftheOMP40antibodies．Accordingly，the remainlngrateSOfPginglValisintheratoralcavitiesatthe COmPletionofthisexperimentwereexaminedbyPCR．Asa result，inthecaseofthegroupthathadbeeninoculatedwith PginglValis，thepresenceofPginglValiswasconfirmedin allsixrats．However，inthecaseofthegrouptowhichthe OMP40antibodyhadbeenadministered，thepresenceofP gingivaliswasnotconfirmedin50utOf6rats（Thble3）．

TART,E 3

DetectlOnOfPgtngtvalu丘omratoralcavitleSbyPCR Table3DetectlOn OfP gtngtvalu丘omrat oralcavitleS bv PCR

Experimental group DetectlOnrate（％）

NotinoculatedwithP．g，Pg＋ Pg＋COntrOlantibody Pg＋anti－r40－kDaOMPantibody Pg only

0／6（0）

5／6（83．3）

1／6（16．7）

6／6（100）

INDUSTRIALAPPLICABILITY

［0126］AsdescribedinExamPles，theantibodybindingto 40－kDaOMPofthepresentinventioninhibitstheaggrega－ tionofPginglValisandpromotesthephagocyticabilityof leukocytes．FurthermOre，the antibodybindingto40－kDa OMPofthepresentinventioncaneliminateP ginglValis from the oralcavity．Accordingly，the antibody or the functionalfragmentthereofofthepresentinventioncanbe e正己ctively usedfor treatlng and diagnoslng Periodontal

diseases．

［0127］Allpublications cited herein areincorporated hereinintheirentirety．ApersonskilledintheartWOuld easilyunderstandthatvariousmodificationsandchangesof

US2007／0036734Al Feb．15，2007

13

thepresentinventionarefeasiblewithintheteclmicalidea andthescopeoftheinventionasdisclosedintheattached

Claims．Thepresentinventionisintendedtoinclude such modificationsandchanges．

SE（⊇UEl寸CE L工ST工NG

＜160＞ NUHBER OF SE（⊇ 工Dl寸OS：2

＜210＞ SE（⊇ 工Dl寸01

＜211＞ LEl寸GTH：2008

＜212＞ TYpE：Dl寸A

＜213＞ORGAN＝SM：PorphYrOmOnaS glnglValis

＜400＞ SE（⊇UEl寸CE：1

gaattccaac cgtggaaagg ggagatagag cggcgcaaca acggatcgat cattgcgctc 60

gaaagcggaa cggcctatgc ctatgccctg aataatctcc aaagccgtgg acgcttcttc 120

atctctccgc aggaggaggt ctatgccggt caggtcgtgg gcgagcacac gaaggagggc 180

gatctctgcg tcaacgtctg caagagcaaa aagcttacca atatgcgtgc ttccggtagc 240

gatgataagg tgtcactggc tccaccggtg gtattcagcc tcgaagatgc tttggagtat 300

atcaagtatg acgagtatgt ggaggtgacg ccgaaaagca tgcgcatgcg taaaattatc 360

CtCgaCgaga CagaaCgtaa aCgaCaagga CgataaCaga ggCaaagtat ttgCagattt 420

CatCtatCta aCgtattttg gtgCCCgaaa taCggaaaat CaaaCaaaat gtttCaCtaa 480

Caaaaaagat aaaCgaaCat gaaaagatta ttaCtCtCtg CtgCtatCCt aagtagtatg 540

gctttgttta atgtcaatgc acaagagttg aaaacctctg ctgacatgaa aggttctttt 600

aagaagaatg tggtattgga ggtatttact gccgaatggt gcggttactg tccaggtggt 660

aaagagcgca ttgcaaaagc aattgaaatg ttggatgatg aatataagga gcgtgttttt 720

CagaCatttg ttCattataa tgatgggatC tCaaaaaaat ggCCtCgtgt tggCCaaCtt 780

ttcattgcat tggatcaaac attgggcatt ccgggttttc cgactttttc agtttgccgt 840

atggagaaaa aaggtgaaaa tctttcaata ggtgctccaa tagcaattaa aaataagatt 900

atgaaaggtt ttggtgatgg tacagcccct gcagaggtaa accttaaatt gaccaaaggt 960

gcaacaccgg aagatgtatg tacagctaca tttactggta aagtcgatgc agacctcata lO20

gggaaacctc ttatgttgac tgcatatgta ttgaaaaaca atatgaagcc tattaatccg lO80

Caaaatggag Ctggggatgg atatCtCCaC CaaCataCtg tgttaatgat tCtCtCCaCa l140

gatgtaaaag gagacgcttt aaatattgca gccgatggaa gttttaccat caagaaagaa 1200

tttaagttgg atggctttga aataaaagat acagatgttc ttgctttcgt acaccatcca 1260

atgtccaatg cggaaaacca ttctattatc aatgccgggc aagaaagcct tgataaagca 1320

gagcctacag ctacagaaca aattgttgct accccctctg tcaaagcata tgttcagaat 1380

ggcaaaattg ttgtagagga agagtattcc aagatggaag tattcaatgc aactggtcaa 1440

CttgtCaaaa atgaatCCCt tgtCCCCggt gtCtatgttg tCCgtataaC ggCaaaCggt 1500

gtaatgcatt tccttaaagt cttagttcct tgatttattg agctaagatt taaacgaaaa 1560

CtgCgCCttC ttaatgtttC ataagaaggC gCagtttCCt tttttCCtCa ttCCattCtC 1620

CgggtggtCg tCgaagggga CtgCCtgtCa tCtttCaaaC gaggaatatC agCtCCCCtC 1680

acccaacacg atgtccacaa atccatgttc atagcctatt tgagaccttt gaaaaaagag 1740

aaggtttctt atatctatta tctataaagg cactaaaaat cagtcgattg acaataaaat 1800

US2007／0036734Al Feb．15，2007 14

＿COntinued

CtCCaagaaa ataCCatgCg aCagaCtCaC tatatagatg CggtCaatgg tCtgatatat 1860

CgaggCtgtt gtggatggag gatagtaCaC atagttttgC aCCtgaaaCa aaCgttCata 1920

CtaaaCtaaa aaCaaaagCa ttatgaCtCC tatCCtgaaC aCCgttttCC CCgagttCaa 1980

2008 actcaatgcc tatcacaatg gcgaattc

＜210＞ SE（⊇ 工Dl寸0 2

＜211＞ LEl寸GTH： 344

＜212＞ TYpE：pRT

＜213＞ ORGAN＝SM：PorphYrOmOnaS glnglValis

＜400＞ SE（⊇UEl寸CE：2

Met LYS Arg Leu Leu Leu Ser Ala Ala ＝le Leu Ser Ser Met Ala Leu

1 5 10 15

Phe Asn ValAsn Ala Gln Glu Leu LYS Thr Ser Ala Asp Met LYS GIY

20 25 30

Ser Phe LYS LYS Asn ValValLeu Glu ValPhe Thr Ala Glu Trp CYS

35 40 45

GIY TYr CYS Pro GIY GIY LYS Glu Arg ＝le Ala LYS Ala ＝le Glu Met 50 55 60

Leu Asp Asp Glu Tyr Lys Glu Arg Val Phe Gln Thr Phe Val His Tyr

65 70 75 80

Asn Asp GIY ＝le Ser LYS LYS Trp Pro Arg ValGIY Gln Leu Phe ＝le 85 90 95

Ala Leu Asp Gln Thr Leu GIY ＝le Pro GIY Phe Pro Thr Phe Ser Va1

100 105 110

CYS Arg Met Glu LYS LYS GIY Glu Asn Leu Ser ＝le GIY Ala Pro ＝le l15 120 125

Ala ＝le LYS Asn LYS ＝le Met LYS GIY Phe GIY Asp GIY Thr Ala Pr0 130 135 140

Ala Glu Val Asn Leu Lys Leu Thr Lys Gly Ala Thr Pro Glu Asp Val

145 150 155 160

CYS Thr Ala Thr Phe Thr GIY LYS ValAsp Ala Asp Leu ＝le GIY LYS

165 170 175

Pro Leu Met Leu Thr Ala TYr ValLeu LYS Asn Asn Met LYS Pro ＝le

180 185 190

Asn Pro Gln Asn GIY Ala GIY Asp GIY TYr Leu His Gln His Thr Va1

195 200 205

Leu Met ＝le Leu Ser Thr Asp ValLYS GIY Asp Ala Leu Asn ＝le Ala 210 215 220

Ala Asp GIY Ser Phe Thr ＝le LYS LYS Glu Phe LYS Leu Asp GIY Phe

225 230 235 240

Glu ＝le LYS Asp Thr Asp ValLeu Ala Phe ValHis His pro Met Ser

245 250 255

Asn Ala Glu Asn His ser ＝le ＝le Asn Ala GIY Gln Glu Ser Leu Asp 260 265 270

LYS Ala Glu Pro Thr Ala Thr Glu Gln ＝le ValAla Thr Pro Ser Va1

275 280 285

LYS Ala TYr ValGln Asn GIY LYS ＝le ValValGlu Glu Glu TYr Ser

290 295 300

LYS Met Glu ValPhe Asn Ala Thr GIY Gln Leu ValLYS Asn Glu Ser

305 310 315 320

US2007／0036734Al Feb．15，2007

15

＿COntinued

Leu ValPro GIY ValTYr ValValArg ＝le Thr Ala Asn GIY ValMet

325 330 335

His phe Leu LYS ValLeu ValPr0

340

1．Anantibodybindingto40－kDaOMPorafunctional fragmentthereof，WhichhasactivltyOfinhibitingthebind－ 1ngOfheminto40－kDaOMP．

2．Anantibodybindingto40－kDaOMPorafunctional fragmentthereof，Whichhas（1）activityofinhibitingthe COaggregationofPgingivalisand（2）activityofpromoting humanneutrophilicphagocytosis． 3．Anantibodybindingto40－kDaOMPorafunctional

fragmentthereof，Whichhas（1）activityofinhibitingthe COaggregationofPgingivalisand（2）activityofinhibiting thebindingofheminto40－kDaOMP．

4．Anantibodybindingto40－kDaOMPorafunctional fragmentthereof，Whichhas（1）activityofpromotinghuman neutrophilicphagocytosisand（2）activityofinhibitingthe bindingofheminto40－kDaOMP．

5．Anantibodybindingto40－kDaOMPorafunctional fragmentthereof，Whichhas（1）activityofinhibitingthe COaggregation ofP gingivalis，（2）activityofpromoting humanneutrophilicphagocytosis，and（3）activityofinhib－ itlngthebindingofheminto40－kDaOMP．

6．Theantibodyorthefunctionalfragmentthereofaccord－ 1ngtOanyOneOfclaims2，3，and5，Whereinthecoaggre－ gationofPginglValisiscoaggregationofPginglValisand dc～∫〝0〝ぴCe∫V∫∫CO∫〟∫．

7．Anantibodybindingto40－KDaOMPorafunctional fragmentthereof，Whichhasactivityofsuppressingalveolar boneresorptlOn．

8．Theantibodyorthefunctionalfragmentthereofaccord－ 1ngtOanyOneOfclaimslto7，Whereintheantibodyisa humanantibody．

9．Theantibodyorthefunctionalfragmentthereofaccord－ 1ngtO anyOneOfclaimslto8，Whichisproducedbya mouse－mOuSehybridoma．

10．The antibody or thefunctionalfragment thereof accordingtoanyoneofclaimslto9，Whereintheantibody isamonoclonalantibody．

11．The antibody or thefunctionalfragment thereof accordingtoanyoneofclaimsltolO，Whichcovalentlyor non－COValentlybindstoatherapeuticagent．

12．The antibody or thefunctionalfragment thereof according to claimll，Wherein the therapeutic agentis Selectedfromantibioticsorantibacterialagents．

13．The antibody or thefunctionalfragment thereof accordingtoclaim12，Whereintheantibioticortheantibac－ terialagentistetracyclineorminocycline．

14．The antibody or thefunctionalfragment thereof accordingtoanyoneofclaimslto13，Whereintheantibody ClassisIgG．

15．The antibody or thefunctionalfragment thereof accordingtoclaim14，WhereinIgGisIgGl．

16．The antibody or thefunctionalfragment thereof accordingtoanyoneofclaimslto13，Whereintheantibody ClassisIgA．

17．The antibody or thefunctionalfragment thereof accordingtoanyoneofclaimslto16，Whereintheamino acidsequenceofaheavychainconstantreglOnisaltered． 18．Anantibodybindingto40－kDaOMPorafunctional

fragmentthereof，Whichisproducedbyahybridomah13－17 （accessionNo．FERMBP－8325）． 19．Anantibodybindingto40－kDaOMPorafunctional

fragmentthereof，WhichcompnsesvariablereglOnS Ofan antibodythatisproducedbyahybridomah13－17（accession No．FERMBP－8325）．

20．The antibody or thefunctionalfragment thereof according to claim180r19，Which covalently or non－ COValentlybindstoatherapeuticagent．

21．The antibody or thefunctionalfragment thereof according to claim20，Wherein the therapeutic agentis Selectedfromantibioticsorantibacterialagents．


23．The antibody or thefunctionalfragment thereof according to any one of claims18to22，Wherein the antibodyclassisIgG．


25．The antibody or thefunctionalfragment thereof according to any one of claims18to22，Wherein the antibodyclassisIgA．

26．The antibody or thefunctionalfragment thereof accordingtoanyoneofclaims18to25，Whereintheamino acidsequenceofaheavychainconstantreglOnisaltered．

27．Ahybridomah13－17（accessionNo．FERMBP－8325）． 28．Anantibodybindingto40－kDaOMPorafunctional

fragmentthereof，Whichisproducedbyahybridoma5－89－2 （accessionNo．FERMBP－8323）． 29．Anantibodybindingto40－kDaOMPorafunctional

fragmentthereof，WhichcompnsesvariablereglOnS Ofan antibodythatisproducedbyahybridoma5－89－2（accession No．FERMBP－8323）．


31．The antibody or thefunctionalfragment thereof according to claim30，Wherein the therapeutic agentis Selectedfromantibioticsorantibacterialagents．


33．The antibody or thefunctionalfragment thereof according to any one of claims28to32，Wherein the antibodyclassisIgG．

34．The antibody or thefunctionalfragment thereof accordingtoclaim33whereinIgGisIgGl．

35．The antibody or thefunctionalfragment thereof according to any one of claims28to32，Wherein the antibodyclassisIgA．

US2007／0036734Al Feb．15，2007

16


37．Ahybridoma5－89－2（accessionNo．FERMBP－8323）． 38．Anantibodybindingto40－kDaOMPorafunctional

fragmentthereof，Whichisproducedbyahybridomaa44－1 （accessionNo．FERMBP－8324）． 39．Anantibodybindingto40－kDaOMPorafunctional

fragmentthereof，WhichcomprlSeSVariablereglOnSOfan antibodythatisproducedbyahybridomaa44－1（accession No．FERMBP－8324）；


41．The antibody or thefunctionalfragment thereof according to claim40，Wherein the therapeutic agentis antibioticsorantibacterialagents．


43．The antibody or thefunctionalfragment thereof according to any one ofclaims38to42，Wherein the antibodyclassisIgG．


45．The antibody or thefunctionalfragment thereof according to any one ofclaims38to42，Wherein the antibodyclassisIgA．


47．Ahybridomaa44－1（accessionNo．FERMBP－8324）． 48．Anucleicacid，Whichispossessedbyahybridoma

SelectedfromthegroupconsistlngOfahybridomah13－17 （access享On No・FERM BP－8325），a hybridoma5－89－2 （accessチOnNo・FERMBP－8323），andahybridomaa44－1 （accessモOnNo・FERMBP－8324）andencodesanantibody COntainlngaVariablereglOnOfanantibodyproducedbythe hybridomaorafunctionalfragmentofthesaidantibody．

49．Aproteinencodedbythenucleicacidaccordingto Claim48，Whichis an antibody orafunctionalfragment thereof二

50．Anexpression vector，Which has the nucleic acid accordingtoclaim48．

51．Ahost，Whichhastheexpressionvectoraccordingto claim50．

52．Thehostaccordingtoclaim51，Whichisselectedfrom thegroupconsistlngOfEscherichiacoli，yeaStCells，1nSeCt Cells，mammaliancells，Plantcells，andmanlmals．

53．A methodfor producing an antibody binding to 40－kDaOMIミwhichcomprlSeSisolatlngagenethatencodes

an antibody binding to40－kDa OMPfrom a hybridoma SelectedfromthegroupconsistlngOfahybridomah13－17 （access享On No・FERM BP－8325），a hybridoma5－89－2 （accesslOnNo．FERMBP－8323），andahybridoma a44－1

（accessionNo．FERMBP－8324），COnStruCtinganexpression VeCtOr COmPnSlng the gene，introducing the expression VeCtOrintoahosttocauseexpressionoftheantibody，and COllectlngtheantibodyfromtheobtainedhost，theculture SuPernatantOfthehost，OrSeCretionfromthehost．

54．Anagentfor suppresslng alveolarboneresorptlOn， Whichcontainsanantibodybindingto40－KDaOMPora functionalfragmentthereofasanactivelngredient．

55．Anagentfbrpreventlng，diagnoslng，OrtreatlngPeri－ Odontaldiseases，Which contains an antibody binding to 40－kDaOMPorafunctionalfragmentthereofasanactive lngredient． 56．Use ofan antibodybindingto40－KDa OMP ora

functionalfragmentthereoffbrproductionofanagentfor SuPPreSSlngalveolarboneresorptlOn．

57．AmethodforsuppresslngalveolarboneresorptlOn， WhichcomprlSeSPreParlnganantibodybindingto40－KDa OMPorafunctionalfragmentthereofandadministerlngthe antibodyorthefragmenttoananimal． 58．Use ofan antibodybindingto40－KDa OMP ora

functionalfragmentthereoffbrproductionofanagentfor PreVentlng，diagnoslng，OrtreatlngPeriodontaldiseases．

59．A method fbr diagnoslng，PreVentlng，Or treatlng Periodontaldiseases，Which compnses prepanng an anti－ body binding to40－KDa OMP or afunctionalfragment thereofandadministerlngtheantibodyorthefragmenttoan animal．

60．Anagentfbrpreventlng，diagnoslng，OrtreatlngPeri－ Odontaldiseases，Whichcontainstheantibodyorthefunc－ tionalfragmentthereofaccordingtoanyoneofclaimslto 26，28to36，38to46，and49asanactivelngredient．

61．Anagentfor suppresslng alveolarboneresorptlOn， Which contains the antibody or thefunctionalfragment thereofaccordingtoanyoneofclaimslto26，28to36，38 to46，and49asanactivelngredient．

62．Useoftheantibodyorthefunctionalfragmentthereof accordingtoanyoneofclaimslto26，28to36，38to46， and49hrproductionofanagentfbrpreventlng，diagnos－ 1ng，OrtreatlngPeriodontaldiseases；

63．Useoftheantibodyorthefunctionalfragmentthereof accordingtoanyoneofclaimslto26，28to36，38to46， and49hrproductionofanagentfbrsuppresslngalveolar boneresorptlOn．

64．A method fbr diagnoslng，PreVentlng，Or treatlng Periodontaldiseases，WhichcomprlSeS PrePanngthe anti－ bodyorthefunctionalfragmentthereofaccordingtoanyone Ofclaimslto26，28to36，38to46，and49andadminis－ tenngtheantibodyorthefragmenttoananimal．

65．AmethodforsuppresslngalveolarboneresorptlOn， Whichcompnsesprepanngtheantibodyorthefunctional fragmentthereofaccordingtoanyoneofclaimslto26，28 to36，38to46，and49andadministerlngtheantibodyorthe fragmenttoananimal．

＊＊＊＊＊

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llllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll · 2013. 5. 27. · PatentApplicationPublication Feb．15，2007 Sheet70f7 US2007／003（i734Al