AU2015264940C1 - Antibodies to OPGL - Google Patents

Abstract

Antibodies that interact with osteoprotegerin ligand (OPGL) are described. Methods of treating osteopenic disorders by administering a pharmaceutically effective amount of antibodies to OPGL are described. Methods of detecting the amount of OPGL in a sample using antibodies to OPGL are described.

Description

ANTIBODIES TO OPGL

The present application is a divisional application of Australian Application No. 2012216429, which is incorporated in its entirety herein by reference.

[001] This application claims priority to U.S. Provisional Application Serial No. 60/301, 172, filed June 26, 2001 , which is incorporated herein by reference for any purpose.

FIELD OF THE INVENTION

[002] The present invention relates to antibodies that bind osteoprotegerin ligand (OPGL). Compositions and methods for the treatment of bone diseases, such as osteoporosis, bone loss from arthritis, Paget's disease, and osteopenia, are also described.

BACKGROUND OF THE INVENTION

[002a] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[003] Bone tissue provides support for the body and includes mineral (including calcium and phosphorous), a matrix of collagenous and noncollagenous proteins, and cells. Living bone tissue exhibits a dynamic equilibrium between formation of bone, which is called deposition, and breakdown of bone, which is called resorption. Three types of cells found in bone, osteocytes, osteoblasts and osteoclasts, are involved in this equilibrium. Osteoblasts promote formation of bone tissue whereas osteoclasts are associated with resorption. Resorption, or the dissolution of bone matrix and mineral, is a fast and efficient process compared to bone formation and can release large amounts of mineral from bone. Osteoclasts are involved in the regulation of the normal remodeling of skeletal tissue and in resorption induced byhnstan e i l by thesc parath-yrcid hQ'oroe in response todresnccntaisoalumnn extacelulrflidsIn contrast,iAnhtonofAresorptonis afunton of calctonin. n addition, metabolites of n ate theesposnessofboneto parath.,yroid hormone anidcactn 0 ostepote na opegeri* a d(0h aPwiciamember afte

TNF familyonf cvoires prootesfo'ainfsec through,bindingt othe

actiatioteceptor or ODAP).steoprotagedn (GO),on theoterhand, nh theofbyequestern P and pireventigOPg

association with ODA Thus heamontof CPGLassociatewthOPDAR

correl th theeqiirumewebon.ede positionandre.rption

[00Q) After 'skeletal1 maturity threamuntof bonein teseto rectst lanceorimbaoanceftone ornaionandboneresorptin a

bone massoccurs afterskeetalmaturtypror tohe fourth decadeB etweenthe

fourthandfifthd ' ecaes, the equyium shif3tsan'd boneesoptindoiates. Thne iae decease inbone massitavan yearsstatearlir

femalesthanootesand isditnclcelrtdatrenpueism emaes(preicipaAy hose ofCaucasan and Asiandescent

[00] Osteopena is a condition relahng general to anyderease

In bonemastobelowormrallievels.Sucaconitinmaarsefrom decrease in e ratof bonesynhess or an easeintherate of bone

d- omonf ommnotofo hO i eo isrt o Sso referred to aspotmenopausaland senile osteoporosis.Thisorof osteoporosisisa consequence of theuniversalloss of bonewithagendisoften

-aesult ofincrseinbone resorpintha normalteofbone formal

Manhtfemales in the UniedState developsymptomatic osteopoos.A

directreltonship existsbe ensteoporossand"theicidenceofhiNpfo neck and ntertrochantercfracture in wnen 45 yarsand oder, dery maes may deveopsymptoatio steopoos isbetweenh ages o0and70,

teooo ayi cerininstancesresul om incraedevels aci of OPGLhus, twold be useful to have molecesthatcan raguatthe actity

QfOP in

tQ0 SeveralfactorshavebeendentiiedwNhmay contrbuteto poemenopausalarndsernieosteoporosis.They ncludeateraoInimone

levelaCCompn igagig and Indequde iumonsumpti ntributedto decreased ntestinaabsorptionofackumarnd otheneras Certaintreatments

have ildehormone therapy or dietary supplemensinatemp to retard the process More recent antresorptieagents suchasphosphonatesan

seeoivestrogenreetrnodifers(SEMshaveemrgedfortepento andtreatment f reducedbonemassThus itmaybeusefutocobinethose

treatments withmolecuesthatcanreguatetheativityofPGL in treating certain osteopenic disorders.

[007a] Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

SUMMARY OF THE INVENTION

[007b] According to a first aspect, the present invention provides an antibody or binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises CDR1, CDR2 and CDR3 of SEQ ID NO: 13 and wherein the light chain comprises an amino acid sequence of SEQ ID NO: 14 wherein the antibody interacts with an osteoprotegerin ligand (OPGL).

[007c] According to a second aspect, the present invention provides a pharmaceutical composition comprising an antibody or binding fragment thereof of the invention and a pharmaceutically acceptable excipient, diluent or carrier.

[007d] According to a third aspect, the present invention provides an isolated composition comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a heavy chain and the second polynucleotide encodes a light chain, and wherein the first and second polynucleotides together encode an antibody or binding fragment thereof of the invention.

[007e] According to a fourth aspect, the present invention provides a method of treating bone loss in a subject comprising administering an antibody or binding fragment thereof of the invention or the pharmaceutical composition of the invention or the composition of the invention.

[007f] According to a fifth aspect, the present invention provides a host cell comprising the composition of the invention.

[007g] According to a sixth aspect, the present invention provides a method of producing an antibody or binding fragment thereof that interacts with osteoprotegerin ligand (OPGL) comprising culturing a host cell of the invention.

A

[007h] According to a seventh aspect, the present invention provides an antibody or binding fragment thereof when produced by the method of the invention.

[007i] According to an eighth aspect, the present invention provides use of an antibody or binding fragment thereof of the invention or a composition of the invention in the preparation of a medicament for treating bone loss.

[008] In certain embodiments, the invention provides for an antibody, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 2 from residue 20 to residue 467 or a fragment thereof, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 4 from residue 21 to residue 235 or a fragment thereof.

[009] In certain embodiments, the invention provides for an antibody, comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 13 or a fragment thereof, and wherein the light chain comprises a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 14 or a fragment thereof.

[010] In certain embodiments, the invention provides for an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 2 from residue 20 to residue 467 or a fragment thereof.

[011] In certain embodiments, the invention provides for an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 13 or a fragment thereof.

[012] In certain embodiments, the invention provides for an antibody comprising a heavy chain and a light chain, wherein the light chain

4a comprises an amino acid sequence as set forth in SEQ ID NO: 4 from residue 21 to residue 235 or a fragment thereof.

[013] In certain embodiments, the invention provides for an antibody comprising a heavy chain and a light chain, wherein the light chain comprises a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 14 or a fragment thereof.

[014] In certain embodiments, the invention provides for an antibody, comprising a heavy chain and a light chain, wherein the heavy chain comprises a first variable region, and wherein the first variable region comprises a sequence that has at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 13, and wherein the light chain comprises a second variable region, and wherein the second variable region comprises a sequence that has at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 14, and wherein the antibody interacts with an osteoprotegerin ligand (OPGL).

[015] In certain embodiments, the first variable region comprises a sequence that has at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 13, and the second variable region comprises a sequence that has at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 14.

[016] In certain embodiments, the first variable region comprises a sequence that has at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 13, and the second variable region comprises a sequence that has at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 14.

[017] In certain embodiments, the invention provides for a heavy chain, comprising an amino acid sequence as set forth in SEQ ID NO:2 from residue 20 to residue 467 or a fragment thereof. In certain embodiments, the invention provides for a heavy chain comprising a variable region and a constant region, wherein the variable region comprises an amino acid sequence as set forth in SEQ ID NO: 13 or a fragment thereof.

[018a] In certain embodiments, the invention provides for a light chain, comprising an amino acid sequence as set forth in SEQ ID NO:4 from residue 21 to residue 235 or a fragment thereof. In certain embodiments, the invention provides for a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 14 or a fragment thereof.

[018b] The present invention further provides an isolated antibody of the present invention, wherein the antibody inhibits binding of osteoprotegerin ligand (OPGL) to an osteoclast differentiation and activation receptor (ODAR).

[019] In certain embodiments of the invention, single chain antibodies are provided. In certain embodiments of the invention, single chain Fv antibodies are provided. In certain embodiments of the invention, Fab antibodies are provided. In certain embodiments of the invention, Fab' antibodies are provided. In certain embodiments of the invention, (Fab')2 antibodies are provided.

[020] In certain embodiments, a pharmaceutical composition comprising an antibody of the invention is provided. In certain embodiments, a pharmaceutical composition comprising a therapeutically effective amount of an antibody to OPGL is provided.

[021] In certain embodiments, a pharmaceutical composition comprises an antibody to OPGL and at least one therapeutic agent selected from a bone morphogenic factor transforminggrowthfator GTCE),an ineiukin 1(I~linhiiton~rierf T, a Th~uAhbito,aslubleTVIA receptor, r n NFr ant bodem cade D2E anybody a paathyroid protein an1anbodryrniedh nereaertenyme tgadna bposh horpone aanat FGF antoofaathyrhr oridcumarer mon e spa etroeamoerb aAPnta nt tarea infammtordrug(NA jaWtihiibitor,Ceatbre*x"-,Voxx_"henr imnunosuopressnmtoteae aeinmi e aseiertaeniig~ secretorleuoc'yteprt ehio(L i,nILinhibitor ntiodyto.,IL ,-$inbioraantibody to[Us-8;anlLiihbtra -Linding protein, an I01!8antodly~an lnterleuki- 1rconveding enzyme(ICE)modulato, a fibobast growt-hfto(FOE, an FOC oultrFP atgoita keratinocyt,,egrwhatrREKOFrelatedmo-, ecu e,KOFmouao atrixmataiproteinaseWMMP) moduator anitcocdesynthaseN moaor8am8odulatroglcocorticoidreceptor aoddiatorogutmter u.22]- ii Inertan emodments ofthe'WvfIona method oftretin anoteoenidisorder is prided, comparing admnsteringaonphamaceuicaly os teoc-cdis order omnprisng admnisteripamcuiacmoiinis provide a bone morphogenic factor, transforming growth factor-P (TGF-p), an interleukin (IL-1) inhibitor, IL-Ira, KineretTM, a TNFa inhibitor, a soluble TNFa receptor, EnbrelTM, an anti TNFa antibody, Remicade TM, a D2E7 antibody, a parathyroid hormone, an analog of a parathyroid hormone, a parathyroid hormone related protein, an analog of a parathyroid hormone related protein, a prostaglandin, a bisphosphonate, an alendronate, fluoride, calcium, a non-steroidal antiinflammatory drug (NSAID), a COX-2 inhibitor, CelebrexT M, VioxxTM;an immunosuppressant, methotrexate, leflunomide, a serine protease inhibitor, a secretory leukocyte protease inhibitor (SLPI), an IL-6 inhibitor, an antibody to IL6, an IL-8 inhibitor, an antibody to IL-8, an IL-i8 inhibitor, an IL-18 binding protein, an IL-18 antibody, an Interleukin-1 converting enzyme (ICE) modulator, a fibroblast growth factor (FGF), an FGF modulator, a PAF antagonist, a keratinocyte growth factor (KGF), a KGF-related molecule, a KGF modulator; a matrix metalloproteinase (MMP) modulator, a nitric oxide synthase (NOS) modulator, a modulator of glucocorticoid receptor, a modulator of glutamate receptor, a modulator of lipopolysaccharide (LPS) levels, a noradrenaline, a noradrenaline mimetic, and a noradrenaline modulator.

[022a] In certain embodiments of the invention, a method of treating an osteopenic disorder is provided, comprising administering a pharmaceutically effective amount of an antibody. In certain embodiments, a method of treating an osteopenic disorder comprising administering a pharmaceutical composition is provided.

[0022b] The present invention further provides a method of treating bone loss in a subject comprising administering an antibody or binding fragment thereof according to the present invention, or a pharmaceutical composition according to the present invention or a composition according to the present invention.

[022c] The present invention further provides a method of treating bone loss in a subject wherein the bone loss is associated with at least one condition selected from osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, osteopenia, osteonecrosis, an inflammatory condition, an autoimmune condition, rheumatoid arthritis, and cancer.

[022d] The present invention further provides a method of treating bone loss in a subject wherein the subject is a human.

[022e] The present invention further provides a method of treating bone loss in a subject wherein the bone loss is associated with an inflammatory condition, and wherein the method further comprises administering at least one additional therapeutic agent selected from an interleukin-1 (IL-1) inhibitor, IL-1ra, anakinra, a TNFa inhibitor, a soluble TNFa receptor,

7a etanercept, an anti-TNFa antibody, infliximab, a D2E7 antibody, a non-steroidal anti inflammatory drug (NSAID), a COX-2 inhibitor, celecoxib, rofecoxib, and leflunomide.

[022f] The present invention further provides a method of treating bone loss in a subject, wherein the bone loss is associated with an autoimmune condition, and wherein the method further comprises administering at least one additional therapeutic agent selected from an interleukin-1 (IL-1) inhibitor, IL ra, anakinra, a TNFa inhibitor, a soluble TNFa receptor, etanercept, an anti-TNFa antibody, infliximab, a D2E7 antibody, methotrexate, a soluble form of CTLA4, and a modulator of glucocorticoid receptor.

[022g] The present invention further provides a method of treating bone loss in a subject, wherein the bone loss is associated with rheumatoid arthritis, and wherein the method further comprises administering at least one additional therapeutic agent selected from an interleukin-1 (IL-1) inhibitor, IL-1ra, anakinra, a TNFa inhibitor, a soluble TNFa receptor, etanercept, an anti-TNFa antibody, infliximab, a D2E7 antibody, a non-steroidal anti inflammatory drug (NSAID), a COX-2 inhibitor, celecoxib, rofecoxib, leflunomide, methotrexate, a soluble form of CTLA4, and a modulator of glucocorticoid receptor.

[022h] The present invention further provides a method of treating bone loss in a subject, wherein the bone loss is associated with cancer, and wherein the method further comprises administering at least one additional therapeutic agent selected from a keratinocyte growth factor (KGF), a KGF related molecule, a KGF modulator, an anti-Her2 antibody, an anti CDC20 antibody, and an anti-EGFR antibody, and a PAF antagonist.

[022i] The present invention further provides a method of treating bone loss in a subject, wherein the bone loss is associated with cancer, and wherein the method further comprises administering at least one of radiation therapy and chemotherapy.

[022j] The present invention further provides a method of treating bone loss in a subject, wherein the cancer is selected from breast, prostate, thyroid, kidney, lung, esophageal, rectal, bladder, cervical, ovarian, liver, gastrointestinal, multiple myeloma, lymphoma and Hodgkin's Disease.

7b

[023] In certain embodiments, a method of treating bone loss in a subject, wherein the bone loss is associated with an inflammatory condition with attendant bone loss in a patient comprising administering a pharmaceutical composition is provided.

[024] In certain embodiments, a method of treating bone loss in a subject, wherein the bone loss is associated with an autoimmune condition with attendant bone loss in a patient comprising administering a pharmaceutical composition is provided.

[025] In certain embodiments, a method of treating bone loss in a subject, wherein the bone loss is associated with rheumatoid arthritis in a patient, comprising administering a pharmaceutical composition of the invention is provided.

[026] In certain embodiments of the invention, a method of detecting the level of OPGL in a biological sample is provided, comprising contacting the sample with an antibody.

BRIEF DESCRIPTION OF THE FIGURES

[027] Figure 1 shows a cDNA sequence encoding the aOPGL-1 antibody heavy chain (SEQ ID NO: 1).

[028] Figure 2 shows the amino acid sequence of the aOPGL-1 antibody heavy chain (SEQ ID NO: 2).

[029] Figure 3 shows a cDNA sequence encoding the aOPGL-1 antibody light chain (SEQ ID NO: 3).

[030] Figure 4 shows the amino acid sequence of the aOPGL-1 antibody light chain (SEQ ID NO: 4).

1e 5shw cheati diagaofm the OGLkpp light chahnexpression plaridOPGL-1KppalpDSRa19.

[032] Figure shows aschematicdiagramoftheOPGL-1gG2

heavy chain expressionplasid,aOPGL-NgG2!pDSKa19,

[033] Figure 7 showsdose-dependentbindingof OPGLto

OPGLcoptedLAlates.

(034] Figure 8 ows specificbding ofOPGL- 1 to membrane

bound OPGC 0r F gure ses inhibkionof uOPG L-Ib ding toOPL

[035}Figure1sowspedciibidingof aCMG-1toOPOL-

coated EJA ptes

ure1showsdosdependennbtonofosteodlat

[038 Figure 12 shows doseedependent nhbionofOPGLbng oDARby aOPGL-1.

Figure 13 showsthmasr cnetaintmpode afterad Zinsteigasige doseofOPGL- tCynomous*onkeys

{040] igure 14 showsthemeanpercentchangeinserumN-T

concentaionafterdm tenasinge dose Of o nom Monkeys.

[041] Figure 15 shows the mean percent change in urine N-Tx concentrationafter administering a single dose of aCOPGL1 to Cynomolgus

Monkeys -,D4 2- Figure'16 showsamntibo.dypoitvend negatvesr conent ie eproflsafterdinsen a single dose of APGL-t

043 Figure1 showsthe aminoac id sequenceofheaOGL antboyh Ahanvrablereio(QD NO:13), 044I Qiureshowsthe minoa dsequenceoofthecOPaPL antibodyghtca beegion0(SE INO;4 Figure 19showsaCel cubdrepaocess fo producion Of

aOPGQ&

[046] Figure2G0show",stihe sera iupercentchngaftr adminster ang asingldose f OPGL itoCynomolgusmonkeys

047F gure2showsthemeanerun AlkainePhosphatase pecen tchango afradmInISteriga Sinegdoseo0 PO 0oynm us

DETAILED DESCRIPTION OF CE TANPREFERREDEMBODMENTS 041 The section headingsusedhereinreoognz~ra puroses only andareottobeconstkruedasliing the sujectmter described dAreferences tedin this appcation are express imcporaed by

refeenceereiforanyproe f049 Standardtechniques may bemused forrecombant DNA, oligonucloisydeicandtisueandtrdtransformaton (,g, electroporationlpotection). Enzymatic reactionsandpurificationtechniques may be performed according to manufacturersspeciicationsorasconmordy accomplished intheart oras describedhereinThe foregoing techniques and proceduresmaybegeneraperford according conventional methodswil known in the art and as described in various generalandmorespecific references that are citedanddiscussedthroughoutthe present specification.

See eg,Sambrooket al Molecular Cloning: A Laboratory Manual (2d ed, Cold

Spring Harbor Laboratory Press,Cold Spring HarboNY(19)),whichis

incorporatedherein by referencefor anypurpose Unless specific definitions are

provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniquesof,analyticachemistry,syntheticorgaicchemistry,

and medicinaland pharmaceutical chemistry described herein arethosewell

known and commonly used inthe art. Standard techniques maybeusedfor

chemical syntheses, chemical analyses, pharmaceutical preparation, formulation

and delivery, ard treatment opatents,

(OS0j As utilizedinacordanewiththepresent disclosure the following termsunlessotherwiseidicatedshalbeunderstoodtavee

following meanings;

[0511 Theterm Isoatedplynucleotide* as used herin shl neanapoynuceo&de of genamic Aor oD synthe ioriginor some combha thereof whh by ofiso nhe otedadyueotd it not associad wh aDor a POWoof aplYnUceoide inwhichl the solatd polynudeocdei foundund in natne (2) i nkedtapo edeh chtisnot Wnedto innaturet or3does not ocaur natreaspartofalr gersequence

[052] The term islatedprote referred to reinmeans a prote coded NA o inan tA sntheticri nors conaon there which (1)isfree of ate aor e iit wuiormllbef ound, (2) is essentally free ofother prteinstorn the same

Sourc eigfrmthe samspec es( ) expressed by a cefroma different s pecieslor4-) doesnt ocurIn naure.

[0531 Thetverm polypeptd~hsusedhtere-inasageeictermto refeto native prtensor uencesthatheel etions, addtionsandlor

substutiso one ore amo acids of the nesequenceThe trm

paoypeptideaso encompasses 00GL1 (as describedbelw, SEQl NO2 and SEQ IDN140:4), or sequencestfha, ha-'ve deeions,adidiions andi/Or substitutonsofoneormore ino a ofaOPGcAccordingtocetan

emboimetsiheinvntonorsesthiehumranhevcainimuogob molseulrepreseted by FiguLre2 (SEQ M NO:2) aidt he hmnan lght chan imniunogbulin moeculeepresentedby Figure4 (SEorfragmens

oranalogs theref,

[0541 Theterm'natura |ly-o ac cur as uedherein asappliedto

an obetefers to the fact that an object Canbe fodi natue Forxamp

polypeptideo poynuceotidese uencehat is present anorgasminriding viue~htabeisltdrmsourceinnatu"rea hasnotbheen v ,ndhich nent namoded by man inMelaborator orotheiseis naturAlyccnTngo

055 eterm opera ynked abused ereinrefersto

componentsthttwein a e oshipnci

manner,For example, a cntr'lsequenrce o-per-ablylinrked`toacdn sequence isigatd insuch a waythatess thec ngsequence achie vedundeconditionscom=Ppatiblewitvfhteorlsuecs ~056 Thter"contrlsqeneasusehereinreerto poynueodesequence whichnmayeffet the epression and processingof

coding sequencestowhich they areigatedThe nature such conro

sequences may differ depending upon the hOst organisms Accordintocerta

embodimentscorol sequences for prokaotes maynludepromoter,

ibosornalbinding siteandtranscription ternationsequenoccording to certain embdi ments controsquences for eukaryntes maypincudepromote

sequences- canincludeeade r S quen0cesand/orfusin fesequeces

[057I The term"poynuetie asreferredtoherenmeansa

omerfom ofuceoes of aleas 10bases ien certain

emboiments, he bases ay ieboeeotides or derbonueodes or a

mcmifiedformmofethertpeofuc eem includessiemd e stranded forms of DNA,

[0¶The termigouloidrfredoeenncjudes aaturrcjyoccurringwad modifi'ednuloilnetg therynaturaty ocourrigend/onnatrallyccrringolgcnucideliagnes O~qouclotiesarapoynuleoidesbsegenraflcomrisngaength of 20mbasesorfwer n cranembodiments,igonucleoes e 0to basesil length. Incertain embodimenslonueotides are 2 3,4,15

17, 18, 19, or 20to 40bAtes ilet.Odoultdsabesiglstranded. or double strandede fore intensruiono agenem tant

Ooucdeotdesof te inveaon may be sense oransense gnuceotides

[0$9] Theterm naturay occu ring nuieode&'nctsdes

deoxyribonudeotidesand ribonudeotides The ten modified uTeeodes"

tern iomo in e uso neo ages uc as

popoWo stme phosphoodohs esposphooselnoate phosphorodiseienoatehsphoroaiothioate ,phoshorsniadate posphoridae, and thel e See, g LaPianohe et udAcids Res, 14:9081 (1986); Stec etalAm.ChemSo1 06:6077 etNui e84);Stein

Ac Res. 16:3209 (1988); Zon et a Anti-ancer Drug Design 6539 (1991);

2o et aL onudodes and Anaogues Practica Approach pp.67108 F(

EcstinE.,Oird0ves Pess, Oxrd England (1@311);Stec etat US, No.5 15 1 Uhrannand Payman Chem calReviews 90543(1990)the

dislsures of which are hereby incorporated byrefeenceor any pupose. An

ogn e a.dec aninclude abe rdeecion

0) dentty adsimilarty ofrlated ad polypepidescanbe

redlyacuaeby known methods? Suchme 'thodsinlue utre no"0tlmted

14 .

to thsedesc inbed nConputaionl o ar oogLe, Oo UnivrsitPreassNewvYor K(1'l9884;Biocoew ngnfracndeoe Proects.SthW academic ReS Newofr(193 ;

alysis ofSUence Data 1G A adGr r G ds Press, NwvJersey (1994;Sequenevalsiin oleua Bolog,Yvon Heinje,' GuAcaemic fress(1987);euecAnalysi.sPrimer,GribsovMand fDevereux, J"adaStoctonress, OS~~~ NewyYork (1091); an'd adf-atSIAM -Sd t- g i v ea

' fO~iJ Prefrredmthodtodeemiedentty aredesigedtgiv

ea esa chbetweenthe sequences estd. MethdstodetenedentiY

are described npubicl availale omputermprogramPeferedcomut

proram- methods to determineidentitybewnwsequences include, but are to G programpackageincludingGAP(Devereux etNuc

s387 (1984)Gen tiCompterrupUnives Wso MadisonWIBLASTP, BLASN, andPATtltcuca,,JM,!.Blo,, 215 403 (190) TheBLASTX program spub iyaa enomthe Natona~eneror~otehnoogInformation (NCD-l'and othersorce(SLAS Mana4lieaaNBJL/IBethiedaM209:Atchtat'7supura 990n. ti e known mihaterman siortmmayas be usedt

e 0623 Ceraialgnmentcheme aligni n gjtramio acd sequences may resultin them1-atching ofonysoreinfhiwoa sequences, and thissmallaigned regionmayvhave ve-yhgsueciett eventhoughthereisnosignIficant relationshipbetweenthe two fu-ength sequences, Ac~ordingly, in certain embodimentsthe selectedagimentmethod

(GAP program)wiresult in an alignmentthat spans at least 50contiuous

amino acidsofthe targetpolypeptide'

063] For exampleusing the omputeralgorithm GAP(Genetics

Computer Group, University of WisconsinMadison,WI), two polypeptides for

whichthepercentsequenceidentity is to be determinedarealigned foroptimal

matchingof theirrespective amino acids(the matched span",as determinedby

The algorithm)In certain embodiments, a gap opening penalty (which is

calculatedas 3X the average diagonat the "averagediagona" is theaveragef

the diagonal ofthecomparisonmatrix being used: the "diagonal" is the score or

number assigned to each perfect amino acidmatch by theparcularcomparison

matrix) and a gap extension penalty (whih isusualy1/10 mesthe gapopening

penalty as wed as comparison matrixsuchasPAM 250 orLOSUM 62are

used injunction with tealgorithm,incertainembodirents,astandard

com prison matrix(see Dayhoff Pt a A s of ProteinSequenceandStructure, (3)(1978) for the PAM 250 comparisonmatrix; Henikoff et al Proc, Nat. Acad

SlUSA,8:10915-1019 (192)for theBLOSUM62comparisonmatrix)isalso

used by the algorith.

in certain embodiments, the parameters apolypeptide sequence comparison include the folowing:

Algorithm: Needlemanaeta, J. Mo.ABo.4&443-453 (1970); ComparisonimatrixBLOSUM 62 fromHenikoff aL supra(1992); Gap Penalty: 12 GapLength Penalty: 4

Threshold of Similarrty:S

[065J The GAP program maybe usefulwiththe aboveparameters In certain embodiments, the aforementioned parameters are the default

parameters for polypeptide comparIsons (along with no penalty for end gaps) usg the GAP algorithm.

As used herein, the twenty conventionalaminoacidsand theirabbreviationsfollowconventionalusage. See immunologyA Synthesis

(2nd Editn. E.S, Golub and D, R. Gren, Eds, SinuerAssociatesSunderland, Mass(199)),which is incorporatedherein by reference for any purpose.

r e(e.gD-amino acds)of thetwentyconventional aminoacids,

unnaturalamino acids such as aa-isubstitutd amino acids,Naikyamino

acids,ltacticacid, and other unconventional amino acids may also be suitable

componentsforpolypeptides of the presentinventonExamplesof

unconventional amino acids include:4-hydroxyproine, hoarboxyglulamate,a

N,N,N-trimethyllysinee-N-acetyllysine,O0-phosphosertne, N-acetylserine, N

formyimnethionine.>3-methylhistidine5-hydroxylysinecN-mnethylarginineand other sirnar amino acids andiino acids (e.g.4-hydroxyprolineInthe

polypeptide notationusedherein,theleft-handdirectionistheaminoerminal direction and the right-and direction isthe carboxyterminal direction, in

accordance with standardusageandconvention.

[067] Similarly, unless specified otherwisetheleft-handendof

single-stranded polynucdotide sequences is the 5' endtheleft-handdirection of

double-stranded polynucleotide sequences is referredto as the 5'directionThe direction of 5to 3 addition of nascent RNAtransriptts referred sthe transcritiondiretion sequence regions on he DNA tandhavingthesame sequenceastheRNA andwhich are 5 to the 8 end fteRNAr anscript are referedto as upstearnsequenensequenceregionson the Astrand avingthe-same sequenceastheNA and wh hare to the3and ofthe RNA transcript arer referred to as dowstream seuencs

S Conservativeaino acid substituonsmay nompassnon naturallyccuinamino aid residueswhicharetypically incoorated by

cemica peptide snthesis ratherthan by sythesa in biologal systems These

include peptidomimtcsand othereversedorinverted forms ofaminoacid

moieties.

[069J Naturally occurring residuesmay bedivided intoolase

based on common sidechain properties:

1) hydrophobic:norieucine, Met, Ala, VLeu,lle; 2) neutral hydrophilic:ys Ser, Thr, Asn, Gin; 3) acidic: Asp, Glu; 4) basic:His, Lys, Arg; 5) residues that influence chain orientation: Gly,Pro;and 5) aromatic: Trp, Tyr, Phe,

[)701' For example, nononservative substitutionsmayinvolvethe

exchangeofa memberof one of these classes for amember from another class.

Suchsubstitutedresiduesmaybeintroducedinto regions of the human antibody

that arehoologouswth nonhurnan antibodies, orinto the non-homologous

regions of the molecule.

[071 Imaking such changes, according to ertainembodiments,

the hydropathicindex of amIno acids may be considered. Eachamino acid has

been assignedahydropathic index on the basis of itshydrophobcityand charge

characteristics They are;isoleucine (4.5) valine(4.2leucine(+3,);

phenylalanine (+2.8);cysteine/cystine (+25); methionine (+1,9);alanine (+1.); glycine (~.4) threonine (C.)serine (-0.8);tryptophan (-.9); tyrosine (-1.3); proline (-146);histidine (432); glutarmate («3,5); gPutamine (35); aspartate(23.5);

asparagine (-?5); lysine (-39);and arginine(-4.5),

[072] The importance of the hydroathicamino acid index in

conferrnginteractivebiological function a protein is understoodin these at

Kyte etlai, j.IMALBia 157:0-31 (1982), it is knownthat certainamino acids

may be substituted for other aminoacidshaving a similar hydropathic indexor

score and stillretainasimilar biological activity,in makingchangesbased upon

the hydropathic index, in certain embodiments, the subStittion of amino acids

whose hydropathicindices arewithin2 isincluded,incertainembodimes,

those which are within are incudedandin certainembodimentsthose within

±&,5areinclded.

[0731 Rtis also understood inthe art that thesubstitutonoflie

amino adscanbe made effectivelyon the basis ofhydrophiicty particularly

where the biologically functional protein orpepide therebyceatedsintended for useinimruneiogicalemodiments as in the present case. incai

emoodimentsthe greatest calavetrage hydrophilicityofa protein ,as governed by the hyrophciy ofits adaent annoacidscreatesiits ammunogenicity and antigenictyvewithabiologicalproperty ofthecrotein. C741 The itBowing bydropiy vaues have beenasignedto these aino add residues: arginine(+30lysire+30)asparta(+301 glutamate(+30± 1) serine (+0);asparagine (40g2):guamine (+02) glycine

(0);hreonine 4prone(051aane (-05);istidine(-);cysteineF

1.0%niethionine );vae(15); eucne (8);so cine(13 yrosine(

2.3 phenylalanine (-25)ndtryptophn (-34.innmaking changes based upon

similar hydrophiicityvales,incertainembodimentstesubstutiofami

acdswhose hydrophilicityva uesarewithin2 isincludedrincedain

embodi-ents those whicharewiin ±1are included and ceain embodiments, those wthin±0.5 are indded Onemayalsidentify ep opee

tram primary aninoacid sequences on thebasi ofhydrophilicivTheseregIons

are aso referredoa epitopicoreregions

075 Exeni ry aminoacidubstittons are set forhiable

Table 1: Amino AcidSubtitutions Original Exemplary IPrefre Resdue Substitutions Substitutions ASiVal, Leuj, 'Je Va Arkg Lys, GIn,Asn Lys _____Asp __n___ Glin Gin Asp Gir GIn Cys f____Se, Ala Set GIn Asn Asn Giu.__ Aspj Asp ____ I___--------- G__ v Hs Pro, Ala Asn, Gin, Lys, Arg } - --------- Ala ___

Arg ------ lie ----- f Leu, Val, Met, Ala, Leu -_____ Phe, Nordeucine ___ beu Norleucine, le, ile Met Vat MAla Phe Ly,4 ____I DArg, 14Diamo-Ag ______ butyric Acid, Gin, Asn I _______

K._Met ILew, P'te, lie fPhe LeuVat lie, Ala, iLea -Leu

O -- Ala - -[-- Thr, Ala, Cys I Thr Thr I____ Ser___ i_ Se____ r Tp Tyr, P- - -I Tyr Tyr Trp, Phe, Thr, er I Phe Vilie, Met, Leu,4 Phe, Lea _____________ Ala, Norleucine i _____

0O6 A skied artsnanibe abito determine suitable vadantsof the polypeptide assefothherein uing welknowntechniquesIneta ambodinentsr one skiledn the art may identifysuiableareas ofthe moecue that may be changedwithout destroying actviwbytagetingregions not believed to be importantfor activty.In certainembodimentsonecanidetifyrtesdues

and portions of themolecules that are conserved among similar poypeptidles.I certainembodliments, even areas that may beimportant 21T for biologicaltactviyor for structure may be subjecttoconservativeamino adsubsttutionsistbout desroyingthe biologicalactivity orwithout adverselygeffetigthe polypeptide

structure. la Additionally, one skilled in the art can review structure functionstudies identfyingesidus insimnlarpodypeptidesthat are importantfo aciviyorstructure inviewofsauch acom parisonione canpredict the importanceofamino acid residuesfina protein that correspond toamnoac residues which are important for activity or structure in similar proteins.One skiled in the artmay optforchemicallysimilaraminoacidsubstitutionsforsuch predictedimportantaminoacidresidues,

[07a) One skiled in the artcanals analyzethe three-d ensional structureand amino acidsequenceinrelationtothatstructure insimlar

polypeptides.inviewofsuch informationone skilled in the art may predict the

alignmentof amino acid residuesof anantibody withrespect tots three

dimensionalstructure.In certain embodiments,one skled in the art may choose

not to make radical changes to aminoacid residuespredicted to b on the surfaceoftheprotein,sincesuch residues may beinvolved in important

interactions with other molecules, Moreover, one skilledintheartmaygenerate

test Vriats containing a single amino acidsubstitutionateachdesiredamino

acid residue. The variantscan then bescreenedusing activity assays knownto

those skied in the artSuchvariantscouldbe usedtogather information about

suitable variants, For example, if one discoveredthat change to a particular

amino acidresidue rested in destroyed, undesirably reduced,oFunsuitable

activity, variantswithsuch a change may be avoided, notherwords,based on information gathered from such routine experiments, one skilled in the art can

readily determine the amino acids where furthersubstitutionsshould be avoided

eitheraloneor combination withothermutations.

[079] A number of scientific publications havebeendevoted to the

predictionofsecondarystructureSee Moult J, CuOp.inBiotet,74:422 427 (1996, Chou et Sicheristy,13(2):222-245 (1974); ChuAt a,

chemistry, 1 3(2)21222 (1974); hou et a v. Ezyrno Relat. Areas

Moi tbl 47:45-148 (1978) Chouett ReV Bioher 4725-276and Chou tat Bopihys.J, 26a384(197.Moeove computer programsarme currentlyavaiabe toassist withpredictng secondarystucture.Onemrethod of redng secondarystutreisbaseduponhomologymodeiig.FRea l two poypeptidesor proteins which have a sequence identtofgreater an or simrdiy geate than 40% oftenhave simiar stucturaltopoogieserecent

growth of the protein structurlaabase PDB has provided hensrced predictabiiy osecondaryst emncdngtepoanumberoffos dn

ayypbdeS Hcn ei, a e

27 1 247 ( 1 RIhas eenggestedeeeta cwps Bot7 (3 a6(1 hAtthereae amied nm oldsnagen

pypepieorotinandthat oneacri tical numberofsutureshavebeen

resoledstructuralpredictionwilecome drmatcalymoec ate

[080] AdditO rmethodsof predicting condarystrtur eincd

"hreadino one ,Ow0p/stmnsB , sn7S : (1997Y Spet a, c1(199p, polea lie a, c2531164 170 (199 OrCbko e hE m 133146-159(19901G bskov t a,

PRoc.N Acd. Sot 84(1334356-43$8 (1987)) andevoutionaryl g(See H m supra (1999,and Brener supra (1997)

[0811 nsertainemnbodimentsanibody vaintsincude -- 4yc osyatovarantswherinteumberan/otypeo -,fgcoytistea been ateredcomparedtotheamino acid sequencesoftheparentpolpeptde

In certaiembodiments, protenvarants comprisea greater or a esser number ofN-nked gycosylationsites than the aveoieA N-inkedglycoseto sitisharctriariyth-.eequence: An-er o--An-X-Thr, whereithe amino acid residue desigatedasAtnay beaymnairsidue except t

.Thesubs oofa inoacid edue t creaehissequed

apotentanew site rthe addon oanN lnked carbohydrate chain Alternat iel ysubstSitutonsicaeimiate thisseq ence wi remove an exisng

Nikedcar ohydratechainAoprovided isaeamranement ofN nked cohydratechainswhereinaor morelned osyationsesipal

thosethaa natural occuingaremnad andn more N-inke sitesarecretedAddtional preferred antibody adants Incde cysteinevarants

eino or nor ey Tein eesidues aredleedfromior ssi tutedfo

theraminoai (mgser neas compareto theopartamno acid

seuence.Cysteevariants may be usefulwhen bodies mst be refolded inO a iciogicail lycnfratoSuIhas Atetheisoutonofinoubl e inclsionbodies.Cyseinevrintsgenerallyavefsewercsteineresiduethan the naerote and typically havan ae numbeto ininie iteracins

resui fromnupairedcystines,

[082 According tocerinembodimentsaminoacidsubt utor

aretosewhh:()r educedsuscepiii to i reduce b

tooxidato, (3a-ter bndinganityformimplexes ()aie

binding affinitiesand/r4)confer ormodifyotherphysiocochernial orfunction

properties onsuchpolypeptdes. According toeeran embodirents sigleor

mAtipleaminoacidsubsttAutions ineranembodmentsconservave amin adsubsitutions) mayb sae tuarady-ocurrnn sequenceinertain embodiants in the poton of the poypeptidenutsideedmin( forming itermoleuarcwntacs Incerin em dimensa conservaveamino aid gabsttintypicaeymayotsubtantalynethestructurlchaactersticof the parent sequence Age replacementaminoacidshouldnot tend ibreaka hex that occurs i theparent sequence, odisrup other typesosecondary structurethatchaacterzesthe parent sequence).xamea-ecoged poypeptde secondary andtertiary struoear e describedinPoteins, Sucturesand oeuarrncipes(Cr eigtnEWHesemana Comnpany,,N-ew York(1HSNQ;InroductotoentucueC$adnn oze edGr ldand s ,Ne 99 ndhrtoeta Natur'e3 54:,105SE(I191),wNhare eachncrporate-dhierein.- byrfeene ah ete moypeptderagmentasedereinresoa polypepUde tat has anamino-teminalandorcarboxytemnaldeletionIn certainembodiments framentsre at east 5 t457aminoacs longtibe appreciated that in certain embodiments fragments areleast 5,6 10,14 20,50,70,100,50,200 250, 300 350400o r400 arn acdslong

[084] Peptide anaogs are common used n pharmaceutical industry anono-ppde dgswitpropertieanalooustotoseofheemlae

peptide These types of non petidecompound re termed eptidemmeticsuo

pAdv,.Drug Res5:29 ( 6);Veber and

FreidinerTNSp92 (1985) and EvanstalMedChant3r229(987

which areincorportedherein by referencefor a purposeSuchcompound areoften developedwith the d ofcomputerized moecua modeling. Peptide mimeticsthat arestruturaysimilar totherapeutically useful peptides may be used to produce similar therapeutior prophylacti effect.Generally, peptidormimeticsare structuralysimilar to paradigm polypeptide (ie, a poypeptdethathas a biochemical property or pharmacologicalactivity), such as human antibody, but haveoneor morepeptide linkagesoptionallyreplaced by a linkage selected from: -CH 2 NH-H 2 CHa -C -, -CH=OH-(cis and trans),- OH 2 , -CH(OH)0H 2 -, and-OH'2 SO-,bymethodswell knownin the art Systematic substitution ofoneormore aminoacidsof consensus sequence with a D-amino acidof thesame type (e.g,DysineinpiaceofL ysne) may be used in certain embodimentstogeneratemorestablepeptidea

Inaddition, constrained peptides comprising aconsensus sequence or a substantiallyidentical consensus sequencevariationmaybegeneratedby

methodsknowinthe art(Rizoand Gierasch Ann.RvBiocem.1:387(1992)

incorporatedherein by reference foray purpose); for example, by adding

internalcysteineresidues capableof formingintramolecular disulfidebridges

whichcyclizethe peptide.

[85] - "Antibody" or "antibody peptidecs)"'refer toanintact

antibody, or a binding fragment thereof thatcompetes with the intact antibody for

specificbinding.In certain embodiments, binding fragmentsare producedby

recombinant DNA techniquesin certain embodiments, binding fragments are

producedby enzymaticor chemical cleavage of intactantibodiesEinding

28, fragments include, but are notirmded to, FatFab, F(ab)2,Fansinge-chain antibodies he trn "heavy hain" ncudes any,o .ypetldehav sufficient vaiabe region sequence to confer pecmafor anOPGLTheterm !qight chan includes any polypeptidehavingsfienvralegoeunc confeseci yan nhenudesa region domain Qnd three onstantegon domains, C12.adS C.The VHdomanis atthe ain-erminus of thepoepieand the0 H 3 domainis a udIength heavy han and ragments thereof Afulength lighc ac e varabereiodominVL andfacosatrgodoman. 0 L C,Likethhay chainthevariabie region domain of thelightchainist at hen ep pideTh termlight chsedhereinco asses length'ilght chainandfragmentshereof.-ebfragmentm is cmp-risedofn ibht chain and theCH and variae regions Of oneheavy hain Theheavy Ai of a Fa molec'(acann otor a disuidebondwithanotherheavy hAi oleculeAFab' fragmentcontansone eight haiand one heavyharnthat containsmreotheconstantregionbetween he andCI domains such thate nab terchind iuide bond caen e sbete boegynsto form aF (ab')2moeueThevegonomriethevaibeeinfobt theheavy andlightchains, butlAsks the cntant reio nsSnl-hi antbodesre Fvmoeueinwhctheheavand"light Chainvaialregion-s hvbeenconcebya flexibleliner to forma singe plypepida chainwvhich` forms an antigen-binding region. Singlechain antibodies arediscussed indeta in' O 88/01649 and US. Patent Nos. 4,946,778 and5,260,2013

[087) Abivalent antibody other than a "uispecifior

"mutifunctiona antibody, in certain embodiments, typical is under to have eachofitsbindngsitesidental

lAa n body sUbsantiay ihibits dhesionofligandtoa receptor when an excessofanibodyreduces'.the quanttyofreceptorbondo

counterrOcpt0rbyat lat abo 20%, 40%, 50%, 80%,85%,ormo

measredi n an vifho ope t bi dingassay).

loss! Theter 'epiopencludesa- ny po peptide d,*nerminatt capable of specific biding to an im ogbobunor-ceeptori certain embodment, sepiope deteR nat ude challyacieua or moleculessuch as asminas~sgriehispopoy~rufn an~icrtaine. mbodments may have speific treedensinasru'cura ara er n spec ch arAn eptope is region f nti gentha t boundbyanib b ynn ertaiembodmntsanantbdyi

sAd to spefal ybind ana ni heni

embodim bd n ts, i saito ec lbinda a

dissociationconstants1iYicetainbdlr-iments,2whenthe

onstanti s100 Mt andin crainemoiet~hnhdsoitocntn

is5§10nb f09O] Thetem"agent isusedheintodenote achemca compounda mixture of chemicalcompounds, a biobgicarcomore n exa madefromb ioogimateras

09 As used herem the teams abe"orabeled" efers to rtcorpo" nfadtectablemark fe ncoa o adiolabeled amn'noatcid oratahmnto a poypeptide Ofbit oitethatcnan hetdetected bymarkedaWin(emg.,streptavidl ncotiigfuoecnmreor enzymaic

activtythacan bedetec ed byoptcaloometricmethos) tan

emodimens th be l orm ar catheapeutcVdof

label gp ypepides andgy opote are kno inthe art and mybe used

examples of labelsf polypeptidesnc e, butae not cited he w

radiosotopsoradionudides e g. 4C15N 35590Y92Th MY rI 125 1 11florescernabels eg, FITd rhoda mnedanthanidepsphrs enzmatclaelseg>hoseadshpeoxidaseB aacoideseNlucierase alanphosphte),cemiluiescen itnygops pedeemined poypeptide epiopesrecognizedby asecondayreporter(egeuciezippe

pai sequences,bindng sites forsecndaryantbodiesmetal bindingdomans

pope tagspin certain embodientsabek are tached byspaarmsof

ariousleghtreduceptetiltihnrace fC92] The te "bilog m asused hereinncludes but s

not i dto: any quans n d substance fomad ngOngororrm y ng thn.uclvnthingsicldebut arenotr~mitedto humans ,ice,monkeys, rWt,rabbits, and otheranialsSuhsusacsicuebt~tarenoli ido blood0serumsurine' ceflsorans tissues1bobenbonemarrowmpnds and skin

Theterm "osteopenic disorderincludes, but is notlimited to,

osteoporosis,osteopenia, Paget's disease, tic bone metastases, periodontitis,

rheumatoid arthritis, and bone oss due to immobilization In additiotothese

bonedisorders, certain cancersare known to increase osteoclast activity and

induce bone resorption, suchasbast, prostate,andmultiplemyeloma,These

cancers are now known to produce factors thatresult in the over-expression of

OPGL in the bone, andleadtoincreasedosteoclastnumbersandactivity.

094] The term "pharmaceutical agent or drug"as used herein

refers to a chemical compound or composition capable of inducing a desired

therapeuticeffect when property administered to patient

[f95) The term "modulatorasusedherein, isacompoundthat

changesoratersthe activityorfunction of a molecule.For example

modulatoray causean increaseor decrease in the magniude of certain

activityor function of a mecuiecompared to the magnitude of the activity or

functionobserved in the absenceof themodulatorIn certainembodiments,a

modulator isan inhibitor, which decreasesthe magnitudeof at least one activity

orfunction ofamoleculeCertainexemplary activities and funcdons of a

molecule include, butare notlimited to, binding affinity,enzymaticactivity,and

signal transduction, Certain exemplaryinhibitors include,but are not limited to,

proteins, peptides, antibodies, peptibodies, carbohydrates orsmall organic

molecules. Peptibodiesare described,eg Fe in WO01/83525,

[096] As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). In certain embodiments, a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. In certain embodiments, a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition. In certain embodiments, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[097] The term patient includes human and animal subjects. Therefore the present invention provides a method of treating bone loss in a patient that is a human, comprising administering the antibodies of the present invention, or a pharmaceutical composition comprising the same.

[098] In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[099] Osteoprotegerin Ligand (OPGL), a member of the tumor necrosis factor(TNF) family of cytokines, is involved in the formation of osteoclasts. Increased osteoclast activity correlates with a number of osteopenic disordersincluding pos-menopausajosteopriesFagt'sdisease yikne metastases and rhumatodarthritis.hereforereduction inO PGLacty mayresultinadcreaseinosteoclast actvty and mayreducethe sevey of osteopenicdisorders cording tocertan ebodiments of theIvent ion antibodies Rected to OPGL may he used eatosteopenic dsorderinluding by notlnmted tothosementionedabove

[O100 certain embodiments of the presentive tionhereis provided a fully human monoclonal antibody against humanosteoprotegeri

ligand(OPGLIn certain embodiments nucdeotide sequences encoding and amino acidsequenescomprisingtheavyndigtchaininmunogobli

mleculesparticiarly sequences coesponding to the variableregonsre

proved In erinembodiments sequencescorresponding to opementay

determining regions(DRs),specifiallfromO R through0ODR3,are provided According to certain embodiments, ahybdoma celline expressin

such'arniunoglobuinmoleculand monodonalantibody i also provided In

certainembodients ,purfied human monoclonalantibody against humanOGL

isprovided

[01011 The ability to clone and reconstructregabase-siedhuman

iocieatartfiachomosomesIYAesand tointroucethnto the mous germine provdes.an appoachto elucidaingqthe fntoacomponents;of vey large orcudely'mapped oclas welas generating usemodelf human

diseaseFurthiermorethe utzatiOn of suchtecnoloor substiution ofmouse

Aloiwih their human equivalntscouldpro.videuniqueisgtsnoh expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

[0102] An important practical application of such a strategy is the "humanization" of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated offers the opportunity to study the mechanisms underlying programmed expression and assembly of antibodies as well as their role in B-cell development. Furthermore, such a strategy could provide a source for production of fully human monoclonal antibodies (MAbs). In certain embodiments, fully human antibodies are expected to minimize the immunogenic and allergic responses intrinsic to mouse or mouse-derivatized Mabs, and thus, in certain embodiments, increase the efficacy and safety of the administered antibodies. In certain embodiments, fully human antibodies may be used in the treatment of chronic and recurring human diseases, such as osteoporosis, bone loss associated with inflammation, bone loss associated with autoimmunity, and bone loss associated with cancer, which may involve repeated antibody administrations.

[0103] One can engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci in anticipation that such mice would produce human antibodies in the absence of mouse antibodies. Large human Ig fragments may preserve the large variable gene diversity as well as the proper regulation of antibody production and expression. By exploiting the mouse machinery for antibody diversification and selection and the lack of imunologcaltoerancetohuman proteins, thereproducedhumanantibody repertoireithese mausestramnsmay yieldhigh affinity antibodiesagainstany anigen of interest, including humanantigensUsingthehybddomatechnology aniger-specific human MAbs with the desired specificitymaybeproducedand selected.

[0104J f nertainebod mentsnemayuseconstantrensrom wpeciesot-her than human wlongw"itheu nvralrgns)

aturahy Occuring Antibody Structure

05 Naturallyoccuring antibody structuranitstypcaly comnpiteraniEach such ermryialicmoeotolnia pairsfpofyp,-,eptidOclhains' each.parhaving one l-entgtk(nceti embodimentsa 2ou5 kDa and one uiieng". avfchain (inertan embodmentsaout 5070 Ma) The amnoatrnaloton ofeachha

yialy incuds avMaiberegonof abut10c'0to110rmoreamnadd "stat

typcalyis responsible forantigen recognition. The carboxytermninaportn of

each ain typicy defines a constantegion that maybeesponsiblefor

effetor function. Hmanligh thainsaretypicalyclassifiedaskappaand

lambdaight chansHeavychainsaretypicalyclassfied asmdetagara alha, orepslo:n'adefntheani Y'S iSotpeas 1gMRgt3,IgA,Ad g respect ivelygGhasseverasubclassesincludin but notlmtedto g ig02 gGZan d g4,g has subclasseincdn uno li{edtog andlgM12.Ifghkissinfaisubdivideditosuassesincudngbut notlimirted':to

FA andigA2WMn fu!Nen th ghtandheavchains bpical thevadable

andconstantregions arejedb aTegn ou 12 ore inacids, 1thhe heavy chainal n reion oab 0moai ,eg.Fundamentaim ology 7 (Pau , ed 2nd ed. Ravenress

NY( 1989P (incorpatedby referecein itsentiretyforaduposesThe

arleegionso f eachlightheavychan pai yi)yif Orm thantigending

sia.

[00 Tarabl rginstpiallyexhiithsamegenera strueof elaivelyconserved frameworkregions( onedbthre hype

varablregon~alocaleoomlemntsitdetrmiinregionsor CDR&.The CDRSrOm theo has of each pair pcayareagned bythe framewrk

regiornswhihayenable bding to a specificepitopeFron-terminaloC>

terminal botlightand heavychanvaaberegions typcay comprise the

domainsFH,0DMPPt ,0R2, FR., CR3 and FR4.The asthmntwof amin-oacidstochdmiityialinacoaneihdfniino Kabat SequencesoProte noimmunologicalnteest (Nato a tesof

Healthethesda 987 and 190 oChotha&Lee IBo 196:0M917 (1987;Chthata.Na4--ture34888(8)

lSisp cifi-or Bliunctioinantiode

01073 A bispecfic orbifuncionalantibodytypcaly ia an natial

hybridantibodyhaving wodifferentheavy/lightchaipairs andtwodifferent

ending sites. Bispecdfic antibodies may be produced by a variety ofmethods indtsingbutnotlirntedto,fusionofhybridomasorlinkingofFfragmets.

See,e .g, Song&slai&LaohmannCin. ExpImmunol79:315321 (1990),

Kostelny etati rrimuno 148 1547-1553 (1992),

Preparation of Antibodies

(010SI Acori Ktcrtiembodimoen'ts;cranntbde specicady bindingte OPGLaeencompasse the ne in,n cetai embodiments, the antibodies may beprducedbm u aon wthfa ength OPGLsoube forms ofOPGora a g tentte ncaiemboi t

antibodOes of theinvennmay be puldnaor mnoonaa ndorabe recombiantantibodes.Inetarnembodimentantibodiesoftenvetonar humanantiadesprepared forexample imunaonoftrangeni animals capable of producing human aniboes geeforexaplePOTPlshed

AppicationNo.WO 3/1227'

[01091 Incertain embodments;the cormplementaity determining regions(ODRs) ofthe ght and heavy chain variable regions of rOPGLmay be

grafted toramework regions(Rs)fomthe same oranther special

cetieboietthe CORs ofthelight andheavchaiinv'aria ble regionsfI oSPOL~abegratedtoosensushmanasTo creaeonnu hanFRsin certainembodmentsRafomseverahumanheavychainor

lightchainamino acidsequencesareaignetientifaconsensusmnoaci sequnceIncetieboiet hemFefrthe OGL-1 havychinrlight chain are replaced with te Rsfrom-,adifferenthevyChAiNorlightcAinin certainembodiments,rare amino acidsin the FRs ofthe heavy andlightchains of aOPG- are notreplaced, while therest or the FR amino acids arereplaced

Rareamino acids arespecificaio acids that are impositions inwhichthey are

not usuahy found in F certain cs.In embodiments,the graftedvariableregions from cOPGL- maybe used with aconstant region that'sdifferent from the

constant region of aOPOL, Incertainembodiments,thegraftedvariable

regions are part of a single chain Fv antibody, D grafting is described,e n U.S. Patent Ns,180,370, 5,893,762, 5,693,761, 5,059, and 5,530,101,

which are hereby incorporated by referenceforanypurpose

[Co0 According to certain embodients,antibodies ofthe

inventionarepreparedthroughthe utilization of a transgenic mouse that has a

substantial portionof the human antibody producing genomeinsertedbutthat is

rendereddeficient inthe production of endogenous,rr urine,antibodies, Such

mce, thenar capableofproducinghumanimmunogobulinmolecules and antibodies and are deficient in the production of murneimmunoglobuin

molecuiesad antibodies, Technologiesutilizedforachievingthisresultare

disclosed in the patents, appLcations, and references disclosed inthe

specification hereinin certain embodiments, one may employ methods such as those disclosed In POTPublished Application No.WO 98/24893, which is hereby

incorported by reference for any purpose. See also Mendezet atNature

Genetics 15:146-156 (1997), whichis hereby incorporatedby referencefor any

purpose.

CI141 Accordingtocertainembodiments, fully humanronoclon

ntbdespecific forGPGTL are Produced-"asfo., lvws.Tranpsyanicmc OWNeinhumanirruo bineeaemuiewttheanieo inters.LmhdoelsscasR-elnirothmcethat express anibodie

are obta ed. Suchrecoveredcesare fusedwiha myeloidtype celine to

prepare imortalhybridoma celinesand suc hybridna scenes are

screened andse stedtoidentifyhybridoma cedlines that produce aibodies

speitcto the antigen ofinterest, certain embodmentstereoductionf hybridoma celli nethat producesantbdies specificto OPGLis proved

01121 ncet ain embodimentsntbodeofthe e oare produced by bybridmaines AM.G AMOG, AMG 6-5AMG and AMG 7.Incetanebimetsanibodiesoftheinventionarpoueby hybrdorra ins AMO ,AMGTAand AMG6 Icerta nembodensthe

antibodies ofthe invention bind to OPGL itha d sociatio constant(Kd) of

betweenapprmte 0.23 end 0. nM i certain embodimentsofAH inventonethe anibodisbind to OPGwth a Kdofless than 0, n.

(01 131 In Certainemodmet teatboieotheinetoare of thelIg(2isotype,In certanembo ietso6ftheneto~hatbde compose human kappa lght hainand a human gG2 heavy chaiIn ceain

embodiment,teanibo."dies oftheinventionhavebe-enclnefrepresioin;

mammliancels certainembodiments, the variableeg onsfthe antibodies

areaicgateo tantr region oerthan te constantregonfor heii

isotype

10'14J In Certanembodimentsoseratiemodifications tothe

heavy and Ught chanscof oOPGA (andccreponding modifications to the encoding nucleotides) will produce antibodies to OPGL having functionaland

chernical characteristics similar to those of aOPGLI.in contrast, substantial modifications in the functional and/or chemical charactestics of aOPGL-1 may

be accomplished byaectingsubstitutionsintheamirnoacidsequenceofthe

heavy and light chains that differ significantly in their effecton maintaining (a) the

structureof then olecuarbackboneintheareaofthesubstitution,forexample,

as a sheet orhelical conformation(b) the hargeorhdrophobiity ofthe

miecule at the target site, or (cthebulk ofthesidechain.

{0116] For example, conservative aminoacidsubstitutionmay ~nvove asubstitution of anative amino acid residue withsanonnative residue

such thatthere is title or noeffect on the polariy orcharge of theamina acid

residue at that position. Furthermore, anynativeresidue in thepolypeptidemay

also be substitutedwith ahnine, as has beenpreviouslydescribedfor"aanine

scanningg mtageness

[011&JDesired amino acid substitutions~whether conservativeor

non-conserativecan bedetermined byhose sied nthearta theta suc substitutionsare des redIncertainembodimentsarno acidsubstitutionscan

be used toidentify mportantmsdues of aOPG-, to grease or dereas the affinity of theantibodies toOPGL described heri.

01173 Incertnmbodimens ntibodiesofte presentinventon

can be presed in cellnes otherthanyridomaell lesIn certain

$9 emnbodimerssequenes encoding particular antibodies can be used for transoimatOf asitablemamlehsteinodiqoeti modment tnformatnc eby knDettoi poynueoIes io a host cli 'Weiorexample poynucleode na virus (or itoaviralVectoand tansduing ashoceWth thevirus(orvector)orby transfection procedres knowi art,as exempNfed by U Pat Nos 4,399,21. 91 4740 iand459A55

(whichpaternsareherebyincorporated herenby referencefor an purpein nemoimenstetansformatin proedre used may depend upnt otto bernfre ehdfornrdutoofheteroogous poyucieEoides intoa lan e aee known Inthe a and incude, but are not [ited to detrnmediate0d transfection calium phosphate pecipttin,polybrenewa mediatedtransfctionprotopastfusoneecooren-encapsuiltionof-Athe:

. oyudectide() in posomesandd retm cronectonoftheDNA io

p113] Maxmmalan celllines aaiable as hossforexrssoar;e well ntheartand nclbut ae nlm otiedcell

linesavnailable fr hemerican ypedu t ur cinATOC),incudingbu

notlimidtoh nesehamster(ovarCHO) C . ) He cels abyhamster kidney(211K) cels,mokykdecepls (C03!humaheaoclulcrinm

cel(e,Hiep0CA), and number ofotherelienetiebdmns

s levels and produceandibo ieswei ng

propertes.

co01l9J According to certainebdmnsanioisfh ,,vntonareusefulfrdetecingQOPGCinioloicamplesl.neti

embodiments, this allows he den ioo a or tissues which producethe protew.In Certaiembodments,antibodieswhicbindtoO (POL andblocck nteracnwIt offer ending compounds n have herapeIticuse

o nse di e taton adbonersoptionIncai

embodimentsantibodiestOPL ma blocitk OPGL MhtO may result ina blok inthesignal transducnascadend NF-k mdateadtran~scriptinciainAsyfrnaui~N~-ndae

transcriptionactvton usgega ucfferasareporter assa re t thoseskiedin the ait

101201 n certainemblodiments, methodsare provdedftra nga bonedisorder comriing adminserinathe lrapeutically!effectiveamounofenr

bonedisorder compisng adrnnistringahrpetcllefodemunoa -nioy toOPOand anothertherapeuticagent in cetainsuchebodim-ens: Theaditinalherapeut iagentisadministered in aheapeutcallyeffedc nIcran eroments,the bone{sa

n tbodneossincludin gu tnotlnmted to,osteopei aand osteoyssQI

cerainembodiments,treamentithanantibdytoOPSGLis used to:supp-res the rateo)fboeeopinTeeoeincetiebdmnsteteta

be used toreuehrtobneeopinhrther esorionrate is ab;ve normal or toreduce bone resorptiontobeonomaleelinorde compensate for below normal levesof4bone formation, incertain embodiments, antibodies can be tested forbinding to OPGL in the absenceorpresenceofOPG and examined for their abitytoinhibit OPL-mediated ostecstogenesis and/or bone resin.

[0121} Condicns which may be treaedardingtocrt

emibodiments include but are nolimted t thefllowing

Osteoporosisicluding, but not iedtopimary endocrIne ostenoorosi inludingbutntmited tohyperthroidim

hyperparathyroidismCushin's syndrome and acromegaly)hereditary

adcongenit forms ofosteaporos (including bunt united to,

osteogeneSis imperecta, haocytinudiaMenkes syndromeRiey-Day

syndrome and osteoporosis due toimmobiiation of extemties Pagasd seas of bone (csteitisdeforans)n adnts and

Osteomyehtis an infectious esionbone, adingto bone

loss; Hyperoaicemiaincluding, but notimedtoshypercacemia

resultingfro solid tumors nc lung,butnotlimited to breastung and

Mdney and hematologicrdOignaciesinodin, but noti mited to ,mUtiple

myeloma, lymphoma andleukemia),idiopathichyperca&cemia,and hypercalcemiaassociated with hyperthyroidism and renalfunction

disorders

Qsteopenincluding but nolimited to,osteoperia flowing surgeryfoeopeiDahduced by sleroid raion osteopenia

associaedwithdisordersofthesmallandlargintstineanosieopenia

assocatedw v thhonic hepa iandrenaliseases;

oteoremrsisssociaedwittramticinjury Q sOO erse oneceldeathndinabtWtimiedtor smionesascte withGacrsisasos teonechrosisoitatdwthseclanemiaS

ose yt inoastas admaeone a asnoctwhde tcbusrshotsdr

an te onecroat associate owin d thorbnat c he withnr o soItdwtheriodoentldisae,ostk)ncrosisssociatedwith Jc~m oishPtheoalicm ~etatiseandosteonoissoiaewihtendtion

Eeparyritis.b": disorders nceainembodm cents anantbody tOPGi susedn con unction

OPGL nc deb are otlmiod thbone morphogectorsdesnated

hrgrowt hctor(T GF)andTGF- fardily membersaintedeukin1( lb-)inhibitors incuding butnolmtedtoI-rand deriatiesthroand",Kinerep'TFoi btosinclu ing butOnotliitedto, soluble TNF receptors, EnbreTM, anti-TNFa antibodies, RemicadeTM, and D2E7 antibodies; parathyroid hormone and analogs thereof; parathyroid related protein and analogs thereof; E series prostaglandins; bisphosphonates (such as alendronate and others); bone-enhancing minerals such as fluoride and calcium; non-steroidal anti-inflammatory drugs (NSAIDs), including, but not limited to, COX-2 inhibitors, such as CelebrexTM and Vioxx TM ; immunosuppressants, such as methotrexate or leflunomide; serine protease inhibitors, including, but not limited to, secretory leukocyte protease inhibitor (SLPI); IL-6 inhibitors (including, but not limited to, antibodies to IL-6), IL-8 inhibitors (including, but not limited to, antibodies to IL-8); IL-18 inhibitors (including, but not limited to, IL-18 binding protein and IL 18 antibodies); Interleukin-1 converting enzyme (ICE) modulators; fibroblast growth factors FGF-1 to FGF-10 and FGF modulators; PAF antagonists; keratinocyte growth factor (KGF), KGF-related molecules, and KGF modulators; matrix metalloproteinase (MMP) modulators; Nitric oxide synthase (NOS) modulators, including, but not limited to, modulators of inducible NOS; modulators of glucocorticoid receptor; modulators of glutamate receptor; modulators of lipopolysaccharide (LPS) levels; and noradrenaline and modulators and mimetics thereof.

[0123] In certain embodiments, an antibody to OPGL is used with particular therapeutic agents to treat bone loss associated with various inflammatory conditions, bone loss associated with autoimmune conditions, or other conditions with attendant bone loss. In certain embodiments, in view of the condition and the desired level of treatment, two, three, or more agents may be administered. In certain embodiments, such agents may be provided together by inclusion in the same formulation. In certain embodiments, such agents and an antibody to OPGL may be provided together by inclusion in the same formulation. In certain embodiments, such agents may be provided together by inclusion in a treatment kit. In certain embodiments, such agents and an antibody to OPGL may be provided together by inclusion in a treatment kit. In certain embodiments, such agents may be provided separately. In certain embodiments, when administered by gene therapy, the genes encoding protein agents and/or an antibody to OPGL may be included in the same vector. In certain embodiments, the genes encoding protein agents and/or an antibody to OPGL may be under the control of the same promoter region. In certain embodiments, the genes encoding protein agents and/or an antibody to OPGL may be in separate vectors.

[0124] In certain embodiments, the present invention is directed to therapies comprising an antibody to OPGL and at least one interleukin-1 (IL-1) inhibitor, and methods of treatment using such therapies. In certain embodiments, a therapy comprises an antibody to OPGL and an IL-1 inhibitor and at least one additional molecule described herein. In certain embodiments, methods of treatment use IL-1 inhibitors and/or TNF-q inhibitors in conjunction with an antibody to OPGL. In certain embodiments, an antibody to OPGL in combination with IL-1 inhibitors and/or TNF-a inhibitors may be used for treatment of conditions such as bone loss associated with rheumatoid arthritis.

roI2$ Ierieukin4(Il ianant inlaammatorycytokine in t states iaedaor nman diseases andd ian

certain instances, L- manufacedby e the copa oc ne genceainsa dances, L1 isproducedinto fm s

1 01261 Adisease or medicalconitonsonsiered tobe an inteieuin-mediated vsease4if the spontaneo,..usoexermetadseor

edial ondiiassociatedwth elevated evesof u s tsue and/r I c or tissues takentromt he body prodceeevtedevelsofIL

alsoacooi~ebythe followinaditin al',! o cnditions;,(1)ptogia fidings associatedwitthediseaseredcacondtioncanbmiice exprientllina nimals byadiitrtoofIUloprultnfersin o fWand (2) apathlogyi nduced i expeientl anialmodelsofthe disease ormedicalconditioncan benhibited oraboished by eamentwh

agentsthat nhibit the action ofI Incertain embe one or more of the abovconditiosaree tin anl medaeddsaseInerinembodimenfts

three ofthe conditiornsare metinan I-Imediateddisease

01211 A cuteand chronicintedeukn-L -)mediated diseases Include, but arenotliAMttotheol, lowigiacute panreaitismYOtrop lateralslersisALS,or,-Lou~ergaies)Aze~ntdsae

adhei/anrexia, including unut notl di ct fatigue syndrome;Qiostrdiunassociatedllnesses,including, but not limited to, Clostriium-associated diarrhea; coronary conditions and indications, including, but not limited to, congestive heartfailure,coronryrestenosisryocardia infarctionmyocardial dysfunction(.g. related tosepsis),andcoronaryartery bypass graft; cancer,including,but notlimitedtoleukemiasincludng,butnot limited to, multiple myeloma jeukemiaand mylogenous(gAMLadCML), andtumormetastasis;diabefes including, but not limitedto. insun-dependent diabetesendometriosis; fever; fibromyalgia; glomerulonephritisgraftverus hostdiseaseand/ortransplantrejection;heohorragicshock;hyperalgesia; infiammatorybowel disease;inflammatoryconditions ofa ot, including,butnot limitedto, osteoarthritis,psoriaticarhritis, and rheumatoid arthritis;inflammatory eye disease, including, butnotlimited to, those associatedwith,forexample, cornea transpant; ischernia, including, but notlimitedtcerebralachemia

(including,but not limited to, braininjury as a result of, e,g trauma, epilepsy,

hemorrhage orstroke,each ofwhichmay iadtoneurodegeneration);

Kawasaks disease;learningimpairment;lungdiseases(incuding,butnot

limited to, acute respiratorydistress syndrome, or ARDS); multiple sclerosis

myopathies (e.g, muscle protein metabolism, including,butnotlimited tomuscle

protein metabolism in sepsis);neurotoxicity(including, but notlimited to, such condition induced by HV); osteoporosis; pain, incudingbutnotlimitedto,

cancer-relatedpain;Parkinsonsdisease;periodontal disease; preerm labor;

psoriasis; reperfusion injury; septic shock; side effects from radiation therapy; temporalmandibuarjointdisease; sleep disturbance; uveitis; and an iflrniIator condton resulting fromM, strai sramcartilagedamage, ammortopedic surgery, ieAtionortherdisaeprocesses f012j n certain embodiments, an L- inhibitormaybeamyprotein or moC cuecapableof specficlyprevening activation ofcelluaareceptorsto

V!, whio may esufrm anynumbroechane empia mechanIismasinclude,but are notlimteto:dwrgltnILirdcin binding fee -,in erfernwith in dingtoitsreetinerfe ngw

foratinotheI lecetorom lee, associatonof LAIreceprwith IL-1 receptoraccessory protein), dinte firing with modulation of 1 ignalingafter bindg toutsreceptor.

[0129 9ertin intereuki-nhiitorinclde, but are not inted to

ILAlreceptr anagonists, inlIudng, but noti'mtedtoKnrtAIL-Ira, iL-i ra: variants, and rde esEich are o iveIytermedI a pOteins;"

anti-receptormoncolonaanatibodies(seee.g_ EP 623674w hishereby

incorporatedbyreferencefor any purpose); I- bindingpoteinincuding, but notimied tosolble Mreeptors (see,e, U. S.PatNo 549268 uS PatNo 5,488032, and US.Pat5No. 544,937AU ,Pat No 5319,071, and U.S, Pat.',No,.5,180,812,whitahrebYnoprtdrfrneoany

purose anti-morona tibodies'1997;C

9402627,WD9006371.Us.Pat No.4935,343, EP 78,EP 2676 1ad EP

220063,whih areherebyicorporatedby referenceftoranypurpose);i receptoraccessoryproteinsandaniodistheretosee e W 96/2367 and

V/Of37 3whihareherebyincorpratedbyreferencefor any purpose) inhibtors intedeukir1 beta converting enyme(CEorcaspase I(seee, wo 99A46243W9 /47O 4 nWO 99/4714whihareeincopor ae bbuderence any uto n beta production andsecreionte k betaproteasei tsando omp undsad proteis that boki AvA/ synthesaor extraceluiarelase of L-I.

[o 0 Exemp ary L-inhibitoorsaredskeneg.inUSPat, N, 1.74T,444;5 3594325608,0355,843,30:$,59,02; 5566,576':5,89,60; A8690315;5872 9 SA S,40m5,9m5,564;n on(WO) patent application 98121957 9/09323, 91117154 ,94 907,/3273,3/235 98/44940, 9847892, MA93 99/0837, 9 299 04291/7249z

9 /327 3 3,9 8/1768,97/08174,9534326z 64269/364 15; E ur 9 9 1 opeaR

) patent appations53497 and 94795; and French patent applicaton FR

276514Thedisclosuresof all of theaforemetionedrference areby incorporated-- "byrefrencForanly purpose

[0131 tee recetontagoni Ira)isa humanprot e thata"ctsasanaturai hibtoroiiterleuki]n-anisamebeofteLfaiy

Wchncudes cndiL-.CaInreeptrto n i

and varants and dervatves thereof as elas methods of makngand using

the~ardzecixbed inULES,Paet ,75422VVO91/0385;W 9 1"!7184 AU 9173536'Wa-)92/16221;W'O 93/21946;WO 94067;WOIC94121275; FR

2706772; WO 94121235 DE42196,e 9420517; WO 9S22793wO G712828andWOQD99/36541, wich ;.,eanopoaehereinbyreerncFora'ny purpose.Incertan embodimets anLlreceptor antagonist may be ycosyate net cain embodiments an Repo antagonist may be non

223eeformsnofILIr a fndvariandescrbedin

U.S.Pat.No.5175222(the 222 patanQ.ThefisfomcaldLi'inte22

patent ischactrized as a 22-2 KDmlueleon SD PAGE witha

approximate selecthi point of 4, hich tsfrm a`Mono QFPLCoumat

td2md NaC in bfp bfe Hf 7mis

characterized asaw,22-23kW protein, which futesfroma ilonocoQuma483

, amCIadrI a INothl -ua r a ne g yok laethehirdf

chaaterizedas 20 kD protinhceu tesf a Q0oumata43

forY~~~i sitngenes nT that code for naSthenhiiors,cloingTosmeesndial Srp0De nca

vecor~tansorinanldtrWSWetighoeenesin to certai3"nceltPsnd expressingthosegene--,st oprduace the nhiitors. iceaiemboments,ee inssaandiod

substitutons (indiduay orcoectieyreferd toas arantaf)aremade WtiteamnaiseuneofIL-Ira. Incetanmbdmet,nIL-Ir~a

(0134] Incertainemoiet hpres entainven~tioni sdirected to! terapies compisig an anybody to OPGL and a leastoneTNai toand

methodsoftreatmentusingsuch th rapiesI Ceainembodiments aihepy

antibody oOPGL andTNainhbitorand at least one addition

molecueedescribedherein

1 Certaindiseasesandmedicaconditions aremediatedby TNF and sbecatgoriedaPSinlamtoyc ito sAehereinta 7NFamedited diseas"includesbut is notiimdted toCadseaseoedica

conditionthati assocated with elevatedeveis of T1in body flu sortisse

and/orDinhic eS sort isuestaken fAr tebody prducee atedlevel

TNFincutureIn cedain embodimentsad seases aTN mediateddiease

(1)athlogcalindngsssoiatdwihthdisasermeiacon -ditio'ncanbte

ni d ain ias by thead inist nr Uppregationo expressionofTNF and/or(2)ptholog"y Jiune'dinexeietlaiamdl of thedisease omeicacondtfioncabi nhibtezd oraboiishedbytreairnenti wI agents that hibittheanof TN

Certain cuteandc honN eeases n l ude

but are notimted o~chexiaand anrex;cncer icuigbu.totiitet, !eukemi" a chroniJcfaiueydom woonary conditonsandorndcaion-s,

incudig~btnolimtedo~cngetivheatfalur,cornaryteno-, sis, myocadialnfarton yocadadysfunctonncldng butnotlimted to such conditionrelated tosepsis andocrona SeSrysaft depesson

diabet iuding, but notiiMAitet juvenieonset ype dabetesdiabetes

e us aidinslnr resistance (ncudng, butnom dtosuliresistance associatedOWoeiy;noeroi~nontii~nrltdodtos fibroma aand analesi agrftersuspeagsa

inammatory bowe d seasesincluding butnot rimted tohn Csdisease and

clostridium difficiieassocAtddiarrea e nldn u niited to, cerebralischemi, which inc~Ldesbut is notlimited to, braininjuryasaresultof trauma, epiepsy, hmorrhage,and/orstroke;lungdisease,including,but not limited to, adult respiratory distress syndrome, asthma, and pulmonary fibrosis; multiple sclerosis; neuroinflammatory diseases; ocular diseases and conditions, ncludingbut not limited to, cornel transplant,oculardegenerationanduveitis; pain, including, but not limited to,cancer-relatedpain;pancreatitis:periodontal diseases;Ptyriasisrubra pilaris (PRP);prostatitis,.including bacterialendnon bacterialprostatitis, and related conditions; psoriasisandrelatedconditions pulmonaryfibrosis; reperfusion injury; rheumaticdiseases, including,but not limited to, rheumatoid arthritis,ostecarthtis,juvenilearthritis(incudg, but not limited to, juvenile rheumatoidparthritis),seronegatepoyatritisankylosing spondyliNs Reerssyndrome and reactivearthritis tsdhease psriatic arthritis, enteropathic arthritis poymyositis dermatomnyositis sdieoderma systemic erosis, vacuiis eg Kawasskrs d erebra is yeg. disease), vasotiliti, Lyme disease, staphyocccainducesepic)ar iSjgrenssyndome rheumatic fever, risand pajmyaigar heumatia ndgiant e -pyho artertis )seiecshock sideeffectsfromradiationtherapy;systeclupus erythematosus(SLE>;temporalmandibuarjint diseasethyroidiisand tssuie transplantaton and/or inflammatory ndiionagresulingfromstral sprain :Ca-rtilage damage,trauma,orthopedcugryifcto'~gV

Clostridun dffiand related speciesorther diseaseorocess,

on f trice nre btgaine TNFi-Iibtm Nbdimytatea ents onefdwne~iainornhbiinTNF productio)n,bi-ndingfreTINEF:inteferig

52$ withT7NFHndigtoitseeptora df 'erfengih dustonof TNF signing afterblndingto s receptor.The trT inhibtof incudes,but notitd todsobilied TNrceptorsaincudin9,butnotliied to solubleunoos factrrecptoryp (shR I15Called thep55rece0ptor),solubletumor necrosisfactorreceptortype H (also called the p5&etn beI anibodies 0 T ncdingbno imrted oRe-icadeand D27see

U.S Ptents cs382 ad6 56 an de TNFee ANF ('e a egWO 98!24463\tanrceptEnbrel) Avaine inhibitorsofTNFo e enzym ACEand ohrmoees af ctTE y

~0438) xerrplary TNFinibors are desede g aopenn patentappliatonsP30537 P422 33 EP34383ER 39832 412 4 P183-014,ER417 563,ER 43 900 EP 464 533; P 5125 2;5 6

905;ER56928 ; E 607776 desrb theu efluomideo inhibitiofTNF-;E63 210;EP 5427 9: E18439 EP 664 12 P 542 795;ER741707;ER74819; EP R8827 EP$80 70;E848783;EP7 701; EP89988; 550376;ER682714;ER853 06EP 550 376;PP943

615P 939 21E6 614 984 P 0 U.SPaten o536 021 ,9291 ;948338 " 280716Z 5Ss95§53'5,834,435;5 172;530s7s42;

5834 435;05156; 5853077; 5Q590337; 5A12445 95953;5 811261

"631145;5C86312;5866 61; 5641673;"8 §77; 5869511; 5872146

51854,003 56461 557722; 1877200;5877151;$886 10; 586960

5527; 5,8913;1877 780; 5,95548O; 555476; 51554351 994,5t 1090119; 52,320: 5952,1; terrina patent appiatuni ND 9055

WNO91/03553 WO 92/0002.WO 92/13096 W/C92/15221,WO93/0o853,WO

93/21946, WO 9/19777,W CH5/34326, WO 96/28546,V O8/2 298,w

9W30541WO 96/38150 VV 638150 VAC97t820 WC97/155 1WO

97/129, VV/C98/o258b1, WOC9512735,V/C9 6P/11209, V/VC98/3ME9 VA2,V/

9/3936 WO NAME/85 WO 98b9315, 98/42659W3O 93329 We

98/43959, WO 8/45268, 98/47663, W 9633172, /O 620926, VA

.9713 7 974, V973977. %VC9 7479 ,'V9,e$Y`37 ,rvV/C9 8/5166,5,Vv'C)

98/4 3946, VVC)95/04045,V/C8156377, WO97/l2244 ,VC0364,V/

9910363V/C6/533#3'W)9/0449 499/01139, VC)28/56788,We~'O

8(6 7 56, VVC9 8/5383-42,WO2/5 29 4B, VV09 852 9 37, VV09 9/ 02 510, VC -)

7/43250 C1099/06410,V/906042&VWO99/09022,VWO99M0688,VOC

99/07629 WO 979/096, W 9O 74 990604 WO9/388

Japanese patent applcatons 10143 023128 0259140 and10130149

10316570, 11001481, and 127800199i;GWoman apliatwiono 1973521;andJ

Briishapplcatonno2 218 101,23268B81, 224$69W.Thediscosusofall

of themfoxernentioned references arehIerebyinopoaebyreerncfrny

purpose.

[EP9 233438 and ER'4,22339describethe amn oacidand

ofeacacd equensofsolublef a0b NFeceptor ype I(aso known a sTNFR or 3OkOa TNF hbtor) and a solble TNF rceptr pe(as knwnas

393 438and EP422 339 a&-',odecrbemoifefomofsTF-and TNK-l, including but notimited to fragments,. functionalderivatives,andvariants,

FurthermoreEP 393 43 and EP 422 339 describe methods for isolating genes

that code for theinhibitors, cloningthegenes intosuitablevectors,transforming or transfectingthe genes into certain cetypes, andexpressingthegenesto

produce theinhibitors. "A0 sTNFR andTFR are members ofethnervetgrowt

factorYTNreceptor superfamy ofrecetors whichincludesthenevgrowth

factorrecepto(NGF),the B cAl antgen D40, 4 8, the rat cefantigen MPG0X40,e fasntige,andt-he00C27 an-d0030aniesSiht

(1990)Siene24810191023 A conservedfeature ofthat groupoel

sorsis cyster chextraceular gand ndingd an whh cabeiidditfour-repea--tedmrotifsofaboutfor~inoacisthatontain& cysteineresidues at positions that aewecon erved(m al (19)

[01411 EP 93 43 teachesa4kDa TNFhN TorASad a 40kDa

TNF inhibtr ASSwhch are truncated versions ofthe fulengtb recombnant

4kDaTNF nhibior protein.A( and53 have 51 or5amno acids,

respectiveydeleted from theaboxy terminus of the mature protein

[0142] PublishedPC Appiataon NicWO, 96/15describes

truncated forms of sTNFR and sTNFRI that do notcontain the fourth doman

(anno acidresiduesThr 2 7 AsnA of ATN9FWandaminoacidresiduesfro

Thr 7 ofAN ;aportionofthethirddomainaminoacid 12 ofe hFRandamnaidr W46 resduesPol2 Lys 4 -OofsTNFR-IQl);and, optonaly, do not contain a portion of hef stdoma namrinoacid residuesAp%

0yaof s 1Nfm and am& ionidressdue yso Nm Hen embodientsthe runcated sTNFsincdudetheproteinsrepesentedby te formulaR 1 fy ~yi3HandR40 2-ylJThese proteins are

truncated os o NFRandsTFRM, rsecely,

01431 As used herein,-ys yeone or morepoeisheen([13y-LICysiOWiseidues1thnroughn.IQC,3 of sTNR-te

sequence ofwhich isprovided in Figue o 98/011 555; hre

represents a methionytedor nonmethionyatedamegroup of Cys or one or moreamino-erminaaino aidresaduesselacted fom Cys MtoAs'; an wpresenacarboxygoup ys or owner mor ecabxy

terminalaminoac dsd ues selected fro Phe to Le1 l 144) Exempiary truncated sTNFRsofthepresent invention

inclu de,butarnoimited to sTNFP-2 6D/0105 sTNR12W D/010, sTNFR 2,60/N105,sTNFR 2.D/d8sTNFR 3/diTNF 2131d5either metbonyltedonnethionylatedandvardants and drtieVsthero. Cerae xemplar truncatedsTNFR a are deserbedeipubfished

Application No.WOS98O155

f0145 As used hereinRs0ysGysl3 R reprsesone or more proteinS whereincys Oys iiresidues OysSthrough ybof sTNER- thesequence ofh chprovidedinFigure ofWO98/15 55

whereinPrpresents ar ethiony ted ornonmethionytedama grou o

ys 32 Ooe ormore6 mIno ermnaamfinodacidresiduesseectedfrom Cy 3t

Leut andwhereinR4represents acarboxy group ofCys'or one ormore

carboxy-terminal amino aciadresidues selected from Aaao Arg 22

[01461 in certain embodiments, thepresent invention is directedto

therapies comprising an antibody to OPGLandat eastoneserineprotease

inhibitorandmethodsof eatmentsing suctherapies h certain

embodirentsa therapy comprises an anybody toQPG andserineprtease

inhibitorandatleastonoeadddonalmoeule described here

[0147] Endogenousrproteoytic enzymes may degrade invading organisms,antigen-antibdy complexes, an ceain tissueproteins thatareno

longer necessaryor useful. nfective agentsmay introduceadditionalproteolytic

enzymes into the organism, Proteaseinhibitorsnayregulatebothendogenous

andinvadingproteolyticenzymes.

(0148 In ceranembodimentsnaturayoccurngproease inhibitors serveto controindogenousproteasesbylimidigtheirreactionsloca

and tempraly incertainembodiment proteaseinbitorsmayihibit

proteases introducedito theodyby fecve agents c aininstances

tissues thatare particularly pronto proteo.at)tackandinfectioninluding

butnot lmitedt those o the espiratorytract arerichinproteasahbitors.

[0149] rtesenhibitoscomprise appoximately % of the

human asma proteins.Atleast eight inhibhrshave been soatedfom this

source and characteedinthe ieratur.Tese inude, but are notmitedto

apha2macrogoPbuInSapha e),pa protease inhiitor (lphalPipha

1antichymotrypsin (alpha Achy),alphaanticolagenase(alpha AC), and

~nter-alpha-trypsin inhibitor ([alpha-I),

050 10ertin instances aistubance oftheproteaselprotease inhibitorbalancecan lead to pteaseedatedissue desuctioncluding, but not otdoepyeaatrtsgloneruo-nephdis, periodontitis, muscular dystrophy tumornvalsona dvaousoter pathoogca ondionsicetai sitaton,g severeathlgiaproessuhas se-psisoaueuk ia

the amount of freepreolyt enzymes presently increaseuoeeease

10151 Furthermore TinaInstances adianhedreguann nhibtorcapacity of the organismayso cause aeration sithe protease/protease inhibitor balance. Anonmiting example of suchadrmnished

relating bor capaciyisan ahatproIteseinbio deficencywis

correlatedvWith the deveopme of pulmonary ephysema

[0152n certainnstances, serous damage tothe ognis can occuwhesucaberr anonitios arepr P~senulese "ursan be taken toc0otroeprtoyiezmsThemrefr poeaenhiiorhvbeen sought that cAn beadmisteredtoan organismcntdprtolytcenzymes

[0153] Lukocyt elastasedtrypsincatbepsinCGandpancreatic elastaseprenol tnexamr-plsofa classoprotieaseasknown assen

proteases. f0'i543 Incertaininstances, eukcyte elstase when released

extraceiuary degsdes cannotive tissue and othervaluableproteisWhilea normay functioningorganism degradesa certain amount of connective tissue and other proteins, the presenceof anexcesiveamount ofekocytelastase maybe associatedwith various pathological states,luding,butnot limited to, emphyseme and reumatoid arhriti sn certanembodiments tocounteractthe effectsof:Jukoc te e kvwhen iAispresent orsg reaterthad normaa praProea ss ofnmibtorhanbeen soughtwichissec reuc easase . Suc apteasin Ibimaybe usefu ifitnwere capableof n isoateduse red viapriaed fomndisuftiintquaniteatobe r u uf

[015$] Ceateut G oerroteelastaserinhibtnckitsaced n

Sohesser etdat, cid nh bitolof danudocyeuralfO oOfeI

Hmanu oserons: iochem irand Possb lnooial FuncioniM

(ads),raadcWaebr~n.(071 andnrJisnSlvse,nn, 5e.Boc.e G,.6S5J 09(198E3).

[ales] Ance inntacs.rpsnnitiates degrad,-aonofceraini sotogntssesuch as panreaictisue,duringajvariet of acutcondi`ions, mc~uingbut roimitdt,pacr a,trypsinihibtoraybeusefulifit colbe isolated andprepareinapurfied form,,anidinsffii.nquantitiestobe

pharaetia useful,

[01571 CahpiCsnthrrtasrs ntiluotsin certinemodi e~athepsn G7iscaalofderdnavaitof proteinsin" wfo~ncuinthsof thecomplement pSAaPanratc tsisante protease thatmay have arolen pancreatisThus hbtorsforhese potssmyabe fc,

I158 nrtIaAajainem bod im")eni-,t s,ths -ubst epcfctad sensitivity to differentinhibtos ofsehiprfear'e believed toreulfrom

changes n ony a few am noacid residues 3y anagy, i may be possibleto v raycsAsdne protease inhibto swhchhnges a relatively few aminoacids w result in nhibition of differentproteasesnc ertin

embodiments, member of hstassinhibits every seinepotease

0101 Anexemplareinepoteaseinh ssecretory eukocyte protease nhibitor SLPI and fragmentsandanis thereof Exemplary serine

o nclde, uta ae t iiedto,poteseALP

mucousprot-sse inhtor (MPO) human seminpasma bronchialmucusinhhibtr(BQandcevcluuiniio(Utincer tan

mbodimentsaserineproteasei bt so may be LPS modulator See

eg in et at19 8 417 in certain embodiments, ese

rrrieulearwelgsuied for UseiJ.n conditions leadingtobonlosbec sehey arpr;eerent-iallirectedto thecriae

U S.PatNo.4,76,13Q;tU.. Fa-t a.30400and1US, PatNo.5163j27, whichreh p eiin ad en rypupod sCEeT Iheoc

disdosed n he foregongreferences as wllsany vaansor anagues

th~ieeoache ctveyotrmed~'eiertaeniio

[01611 IL-18 is a pro-inflammatory cytokine that was found to induce interferon-y and was previously named interferon gamma inducing factor (GIF). In certain instances, IL-1 has been shown to upregulate IL-18 production, and IL18 induces production of a number of proinflammatory cytokines, including IL-6 and MMP-1. See, e.g., Dinarello et al. (1998), J. Leukocyte Biol. 63: 658-64. In certain instances, caspase I is also important for IL-18 production. Experiments also suggest that TNF-a regulates IL-18 production, and that simultaneous inhibition of TNF-a and IL-18 protects against liver toxicity. See, e.g., Faggioni et al. (2000), PNAS 97: 2367-72.

[0162] IL-18 acts in vivo through a receptor system reminiscent of the IL-1 system. IL-18 interacts with a cell surface receptor (IL-18R), which interacts with an accessory protein (IL 18RAcP). IL-18-mediated signaling proceeds upon formation of the complex of IL-18, IL 18R, and IL-18RAcP. A natural inhibitor for IL-18 is IL-18bp. In certain embodiments, IL 18bp acts as a "decoy receptor" by binding to IL-18 molecules and preventing interaction with IL18R.

[0163] In certain embodiments, the present invention is directed to therapies comprising an antibody to OPGL and at least one IL-18 inhibitor, and methods of treatment using such therapies. In certain embodiments, a therapy comprises an antibody to OPGL and an IL-18 inhibitor and at least one additional molecule described herein. Exemplary conditions that may be treated according to certain embodiments include, but are not limited to, bone loss associated with inflammation, autoimmune diseases, bone loss associated with IL-1 mediated diseases, and bone loss associated with TNF-mediated diseases. Exemplary conditions that may be treated with an antibody to OPGL and at least one IL-18 inhibitor according to certain embodiments include, but are not limited to, the loss of bone and cartilage accompanying arthritis, including, but not limited to rheumatoid arthritis; systemic lupus erythematosus (SLE); graft versus host disease (GvHD); hepatitis; sepsis.

[0164] Exemplary IL-18 inhibitors include, but are not limited to, antibodies that bind to IL-18; antibodies that bind to IL-18R; antibodies that bind to IL-18RAcP; IL-18bp; IL-18R fragments (e.g., a solubilized extracellular domain of the IL-18 receptor); peptides that bind to IL-18 and reduce or prevent its interaction with IL-18R; peptides that bind to IL-18R and reduce or prevent its interaction with IL-18 or with IL-18RAcP; peptides that bind to IL-18RAcP and reduce or prevent its interaction with IL-18R; and small molecules that reduce or prevent IL 18 production or the interaction between any of IL-18, IL-18R, and IL18RAcP.

[0165] Certain IL-18 inhibitors are described, e.g., in US Pat. No. 5,912,324, issued July 14, 1994; EP 0 962 531, published Dec. 8, 1999; EP 712 931, published Nov. 15, 1994; US Pat. No. 5,914,253, issued July 14,1994; WO 97/24441, published July 10, 1997; US Pat. No. 6,060,283, issued May 9, 2000; EP 850 952, published Dec. 26, 1996; EP 864 585, published Sep. 16,1998; WO 98/41232, published Sep. 24, 1998; US Pat. No. 6,054,487, issued April 25, 2000; WO 99/09063, published Aug 14,1997; WO 99/22760, published Nov. 3, 1997; WO 99/37772, published Jan. 23,1998; WO 99/37773, published March 20, 1998; EP 0 974 600, published Jan. 26, 2000; WO 00/12555, published Mar.

,2000; Japanese patentapplicaton JP 111399/94, pubishedOct 31, 197 Israel patentapplicatio iL 1215'-54A,pubisedeb0 8;wrcare inopoaehereinbyrfe*".rence foranpurpose'

[01661 incerainemoimnsaanibody'to OP2 Lm ,aybeuse withaotlAstonefhrapeuticangjenfor nlmainin ceqan embodimnts,an antibodyto' OPOL may beusved withat esoneterpetiagen-tf'oran immunedisorer.Exemplartherapeutiagents "for~lmainnimn disordersc but are not edato,r c rid id bnotlimited

to prednisoione nonsteroidalantnflamatorydrgs~tst'noaludinbut notniedtoyioxygenaetype x adcydooyen eype

notlimiedtoehteaehdoxoirqieciroun ylsoiegodj compounds (such asauranoi, auohkoWalateenartioicsand

Rolipramand Pento i eacr orus 5Siroiusaparyn moChenoliard;d poxygenase hborsicudingbutnomitedto

ZIeutnodult orsof el -(); small cuduatrsof3)a

ntraceular eulesinolved inilammatonpathwayswhereish

intacellar moecues nclde, but are notnimid to,jkIK NF-«, ZAP7O

Cetai exempylarth eapeuf cage tsfinflammcatine descbed

e3

Amgen Inc. Thousand Oaks,GCACertain exemplary therapeutic agents for infammaton and autoimmune diseases include, but are notimited tointeeron

gamma (FN/)modulators; modulatorsofX40/X40L(includingsolubleforms

of x0); modulators of 4-8/41igand (including soluble formsof4-$);

aid modulators of SBceT cell costimultorypathways,including,butnotlimited

to, modulatorsof the receptor igand pairsCD28/T7,C40/C40L,

ICOS/B7RP1, and AGP-ABAFFR(AGP-3 binds tobothTACl and SAFFR

receptors), Certain exemplary modTators ofB c-cellcostimulatorypathways

include, but are not mited to,inhibitors ofCD28, 71, and7,2 (including

soluble forms of F7,1 or 87.2 and double formsof CTLA4, both of which may be

fusedtoa heterologous peptide orprotein whichreducesorprevents

degradation and/or increaseshlf-ife,reducestoxicity,reducesimunogenicit,

orincreasesbiological activity of a therapeutic proteinbyincreasing solubity or

irculatinghalf-life);inhibitors of CD40 and CD40L(including soluble forms of D40 whichrnaiy be fused toa eterologous peptide or proteir);inhibitors of IGOSand B7RPI(includingsoluble formsof1 whichmay refused toa

heterologous peptide or protein) andinhibitorsof AGP-3, TAC and BAFR

(including solublefos of TACI and SAFFR).1 0,B7RP1 andinhibitors

thereof aredescribed,eg,in WOO/46240 AGP-3,TAC and BAFR and

inhibitors thereof are described, e.g, in WO00/47740,WO01/8687,

WVO2/273,AO8/931,and vonulowand Brain(1997)S6cience27S:138- jn67 tcerinaembodiments an antibody to OPGLis usedto treatbonecosinluding butntlimited tobonelossresultngromosteoyt destruction of bon caused by megntornmetatictumors hIcertain embdient aantibodytoOPOIW.'L may beusedttreabonelCjossascte withcancer.Exemplar-ycacesnclude butareoimi ttobreast, prostate, thyrod, kdney ung, esophageas retaWbladder cericaovarian andiver cancers, aswe ascancerfthegatrointetonaltracnertaiembdents anantibdy to OPG may be used toteat bne ossassocatedwth eqg certinheatooqiclmalgnaciesincldin,Ainot'illedt,multipe

010} Intinembodime nantIbod t OPL is admnitered aloneIncertain embodimers, anantibody toOPGLis

administered with atleast one othetheraeutic ageninlding~btnolmted toat least oneoher acerthrapy agentEx le rte -a s il de but are notited to radon thepyand chemotherapy certain

embodment-cheotherapy may involveteamntihoermrofthe followng: antiiroies talF tmxiee oorbci,-furorc other drug-s knowni.n theartin cetainem,-bodime3ints, acancertihernpy age,ntisa

Jutinzinhomon-rleainhormoe(HH) an'tagoitIeti

embodimensa LHRH antagonists a peptideantagons (019 in Acertainembodiments, artLHPHanaonscompris esthe"J

peptide:AN Pe TDAn~ ydraeAs NH2(SEQ IDNO:0Mwhere Nafis 32napthyl)alaninyi4; khais4 chibrophenyv~ataninyl;Pais 3pyflvlaarnn andysPKiNpson propyllysinyl

[0170] in certainembodiments, an LHRH antagonists anLHRH antagonist decapepUde. Certain exemplarydecapeptidesaredescribed, egin

US. Patent No. ,843,901 whichis herein incorporatedby referencefor any

purpose.

[0 171] Exemplary therapeutic antibodies accordingtcertain

embodiments include, hut arenotiited to,rnosemousehumanchimeri,

CDR-grafted, humanized and fuy human antibodies, and synhetic antibodies,

including,but not limited to, thoseselectedby screening antibodybraries

Exemplary antibodies include, but are not limited to, those which bind to cell

surface roteins He2, CDC20 CDC33, mucin-lkeglycoprotein, and epidermnal

growth factor receptor(FR present on tumorcells, and optionally induce a

cytostatic and/or cyttoxic effect on tumor celsdisplaying these proteins.

Exemplary antibodiesalsoinclude HERCEPTIN T (trastuzumab),whichmay be usedtotreat breast cancerandothero-ofcancer,and RITUXANT

(rituximab), ZEVALIN (britumomabtiuxetan),andLYMPHOCIDEh

(epratuzurnabwhich may be used to teat nonodgkirslymphomaand other

formsof ancer.Certain examlaryantodiesalsoincludeERBITUXTM(IM

C225), EXXAR'h lodine31tositumomabandCampath,

10172] in certain embodiments,cancertherapyagents are

polypeptides which selectively induce apoptosis in tumor cellsincluding,but not

limited tothe TNF-related polypeptide TRAILincertain embodiments,an

6aN antibody to OPL ma be admisteredaeastoepriopato concurrent with andsuseuetotreatmentith-ac;anc-er therapy agent Incerain emrdants, an antibody to PGL may beadministered rophyatleyto prevent or mtgate the onsetof bone Lossbymetastatccance cean embodimentsan antibody toODPGLmaylbeadiiseeforthletreatmentof an exitinconitinoboneiossdu'eto met-astss

[01711 incertanembodiment,anatibodlyt oOPG3Lmay beu sed opreventandortreatboneosassoated thmuiplemyom aand to event and orseatthe dlasiselfMutI pmyeornaisa cedei ed

ratmayr esulin m ot yan rmoraitnetan

stancesacinical 'nifesoftatmionu#tplyeiomnais focalboneoss hch

may be due toncreased ostoclastctvaionin Loaiedregons.Many iyIoina patinspresentw th bone lesions Vse byadioogiclanys and

suffer fromskeletapai.in certanntances patents wh myeoma are

susceptibletopathologicaacturesof iwoved bonewhich mayoureither nta us toinjuryI n ca sane Sk AS tat

occurduringrnyeloa noton-,lyleadto boneratuesbut lsodfriyn ccasionay von oheverteb asin $sme

patients aptho ical increase n serum cacium (hyperacemia occurs,and

may causesigifcant problems during diseasetreatment.Incetan 'bn neresoar tn anodyoP s msayb e am initeed to patientytd s tore

bokbonreortionand release of calciu, whichmarce-tL *theriskc fr' Iaturemsarispnldefriy

[0174] In certain instances, myeloma cells do not directly participate in bone destruction, but instead produce extracellular signals that lead to osteoclast differentiation and activation. In certain instances, osteoclasts produce high levels of the cytokine IL-6, particularly when they become activated. IL-6 is a B-cell growth factor, and contributes to the growth of both murine and human myeloma cells in vitro. Myeloma cells may also either directly or indirectly produce OPGL, which may result in local bone lysis surrounding the myeloma cells embedded in bone marrow spaces. In certain instances, the normal osteoclastsadjacent to the myeloma cell in turn produce IL-6, which may lead to local expansion of the tumor cells. Myeloma cells expand in a clonal fashion and may occupy bone spaces that are created by inappropriate bone resorption.

[0175] It has been observed that OPG administration in rodents induces rapid death of the osteoclast population (see, e.g., Lacey et al. (2000) Am. J. Pathol. 157:435-448). A reduction in the number of osteoclasts may counteract the effect of increased IL-6 production by those cells and may therefore affect the growth and survival of myeloma cells within trabecular bone. Thus, in certain embodiments, administration of an antibody to OPGL to a myeloma patient may block the hyper resorption of bone, but may also affect the expansion and survival of the tumor itself.

[0176] B-cells express the receptor for OPGL, ODAR. Myeloma cells also express ODAR, and in addition may produce OPGL. In certain instances, the expression of both OPGL and ODAR in the same cell population may create an autocrine stimulus that may affect survival of the myeloma cell. Thus, in certain embodiments, administration of an antibody to OPGL may reduce tumor cell survival, and may thereby decrease or eliminate the tumor burden seen in myeloma patients.

[0177] In certain embodiments, the invention provides for pharmaceutical compositions comprising a therapeutically effective amount of an antibody to OPGL together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.

[0178] In certain embodiments, the invention provides for pharmaceutical compositions comprising a therapeutically effective amount of an antibody to OPGL and a therapeutically effective amount of at least one additional therapeutic agents, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. In certain embodiments, the at least one additional therapeutic agent is selected from bone morphogenic factors designated BMP-1 through BMP-12; transforming growth factor-p (TGF-P) and TGF-p family members; interleukin-1 (IL-1) inhibitors, including, but not limited to, IL-1ra and derivatives thereof and Kineret TM; TNFa inhibitors, including, but not limited to, a soluble TNFa receptor, EnbreTM, antiTNFa antibodies, Remicade TM, and D2E7 antibody; parathyroid hormone and analogs thereof, parathyroid related protein and analogs thereof; E series prostaglandins; bisphosphonates (such as alendronate and others); boneenhancing minerals such as fluoride and calcium; non-steroidal anti inflammatory drugs (NSAIDs), including COX-2 inhibitors, such as CelebrexTM and VioxxTM; immunosuppressants, such as methotrexate or leflunomide; serine protease inhibitors such as secretorieecyepoeaseinhaistora~)benios (esg antibadies toIL108I -8ihibitos(e.ganibodies toL-8:L-I 8ihbtors (ag, ISbinding protein or L-antibdis) [nterieukincorteingnzyme (E ' modulators'fibroblast grmwitfactorsFRPItoGFGFIandP dulators; PAF antagonists;eratinocyte growthftor(KGF), KF-related moecleoK.GFodultorsmtrx metaioprote inas'ei(MP)miodulatMort Ntri oide synthese(NO5)modulatorsilud ngSmodulators of inducenOS modulators of giucocorticoidrfeceptonmodulatorsofgutaaeecepto; modulatorsofliooyachrd(LPS)leesanrdrea and moduators and miecthere

[017 In cMrain embodimentsacceptable formuationmaterials

preferablyare nontoxic to recipients at the dosages andconcetratons

employed. Inmcetian embodiments ,thepharmaceucacompositi

maycbontainformulaion materisformodiyin maintaining or presevngnfor exanplehe pHosmoNty, viscosity aritycolorisotonicityodorsterity,

stabity, rate odisso ution or reese, adsorptonor penetratiooftie

composiion Incerainem bodiments,suitablWefo ltrateias include",but are notmedto ainoaids(suhas yn utn-ineasagneargin

orysine);antimcrobal; atoxdant (s uchassobccdsodiu.msulitor sodiumhydrogen-ute);bufers(such as boratebirbonateTrisH cit ates

psphatesor oher raacids;buking agents(such asmaagor ycne);

cheatng agents uc e eniam aterasecaid(ETA;compAg

7G agents (such as caffeine,poyvinypyrrolidonebeta-cyclodextrinor hydroxypropy-betacyclodextrin);fwers; mnosaccharides diaccharides; and othercarbohydrates(such as glucose, mannose ordextrins);-proteins (suchas serumalburin,gelatinorimmunoglobulins); coloring, flavoring and diluting agents; emulsifyingagents; hydrophilic polymers(such aspolyvinyipyrroldone); lowmolecularweightpolypeptides;saltdormingcounterions(suchassodium); preservatives (such as benzalkonium chloride, benzoacid, salicylicacid, thimieros-al,phenethyl alcohornethylparaben, propylparaben, chorhexidine, sorbic acid or hydrogen peroxide); solvents (suchasglycerin,propyleneglycolor polyethylene glycol); sugaralcohols(such as mannitolor sorbitol);suspending agents;surfactants or weting agents (suchas pluronics, PEGsorbitanesters, polysorbates such as polysorbate 20,polsorbate$ 0,triton,tromethaine, lecithin, cholesterol,tlyoxpal; stability enhancing agents (such as sucrose or sorbitol); toniity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride,nannitol sorbito); delivery vehicles;dluenits excipients and/or pharmaceutical adjuvants.,(Remington'sPhanmceutica/ Sc/encesR Edition, F8 AR.C ennaro, dMack PublishingCompany(1990).

[01811 uncertain embodiments, anantibodytOPGLand/ora

therapeuticmolecue is linked to ahalfife extendingvehicleknownintheart

Suchvehicles include, but aren't limitedto,the Fc domain, polyethylene glycol, and dextran.Such vehicles are described, in UApIiction Serial No.

09/428,082 and published POT Application No. WO 99/26044,whicharehereby

incorporated by referencefor any purpose,

L01821 in certain embodimentsthe optimalpharmaceuticat compositionwill be determined by one asked inthe art depending upon, for

exampie,the intendedroute ofadinistration deiveyformt anddesired

dosage. See, forexampleRemngton'sPharmaceutcalSciences, suprIn

certain embodiments, such compositionsmay influence thephysicalstate,

stability rateof in virorelease and rate of invivoclearanceoftheantibodiesof

the invention.

[Ol831 n certain embodiments, the primary vehicle or cater in a pharaceuticalcomposItion may eeitheraqueous or non-aqueous innature,

For example, in certain embodiments, a suitable vehicle or caermaybe water

forr nction,physiological salinesolutionorartificial cerebrospinalfluid, possibly

suppementedwithother materialscommon in compostons forparenteral

administration Incertain embodients, neralbuffered saline or salinemixed

with serum albumin are further exemplary vehiles. Incertainembodiments,

pharmaceutical compositions comprise Ths bufferof aboutpH0 ,oracetate buffer ofaboutpH4,0-,5,which may further include sorbitol or a suitable

substitute therefor,In certain embodimnens a composition comprising an antibody to OPGL with or without at leastone additionaltherapeuticagents, may

be prepared forstorage bymixing the selected compositionhavingthedesired

degree of purity with optional frmulation agents(emingon'shamaceuical

Sciences, supra)in the form of ajyophitized cakeoranaqueoussolution

Further, in certainembodiments, a composition comprising an antibody OPG1 to

72, with or without at least one additional therapeutic agents, may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0184] In certain embodiments, the pharmaceutical compositions of the invention can be selected for parenteral delivery. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

[0185] In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

[0186] In certain embodiments, when parenteral administration is contemplated, a therapeutic composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired antibody to OPGL, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In certain embodiments, a vehicle for parenteral injection is sterile distilled water in which the antibody to OPGL, with or without at least one additional therapeutic agent, is formulated as a sterile, isotonic solution, properly preserved. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide for the controlled or sustained release of the product which may then be delivered via a depot injection. In certain embodiments, hyaluronic acid may also be used, and may have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices may be used to introduce the desired molecule.

[0190] Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving antibodies to OPGL, with or without at least one additional therapeutic agents, in sustained- or controlled delivery formulations. In certain embodiments, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, PCT Application No. PCT/US93/00829 which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments sustained-releaseapreparations may include semipermeable polymer matrices in the form of shaped articles, e, films, ormicrocapsues

Sustainedrelease matrices may include polyesters,hydrogelspolylaotides(U.S,

3,77319 and EP 058,481), copolyners ofLglutamic acid and gamma hy glutamate (Sidmanet l,Bopoytmers, 22:547-55(1983), poly(-hydroxyethyp

methaoryate) (Langer et at, I Biome. Mater Res 15:16-277 (1981)and

Langer,GChem.,Tech,12:98-105 (1982)), ethylene vinyl acetate(Langer etatL supra) or poy---3-hydroxybutyrioacid(EP 33,988 Incertain emodiments,

sustainedreleaseompositions may also include liposomes, which canbe

prepared by anyof several methods known in the art Seeage. ppsteinaetat ProcNatL Acad.SciUSA. 82:3688-3692 (1985): EP 036,676 E08,046and

EP W494

[01911 The pharmaceutcalcomposition to based forVVQ

administrationtypicailyistere i ctan embodiments thismaybe

accomplished by filtftratithughsterlefltraionmembranesincertain

embodimentswhere the compositions yaphlized steilzationusngthis

method may be conduted either por t or fol ingyophilization and reconstitutiornncertan embodimentshecomposionor paeneal

administration be storedinlyophilized form orinasoluionIn certan ernoodhnts parenteralcopiinonIOsgeneraly are placedintocoaCianer havingoasterile access port forexampean intravenous slutionbag or vaa -m. in ,, -7.e having astopper pierceable by hypodermic inectionneedle i021 ineinembodments o thepharmaceudea compositionhas been fbmuaitedR may be oedin steie vials asuon, suspension, get emulsion, solid, or as a dehydratedkorlyophilizepowderin certainembodiments, such formuations maybe stored eitherina readyto-use formorinaform (e.g ophilized) that's reconstitutedprior toadministration.

[01g3] In certain embodiments,thepresentinvention is directedto kits for producing asingle-dose administration unitIn certain embodiments, the kits may each contain both a first container having a dried proteinand second

container having an aqueousformulation, Incertain embodiments ofthis

invention,kits containing single andmulti-chamberedpre-led syringes (g,

liquid syringes and lyosyringes) are included,

[0194 In certain embodfrents, the effective amount of a

pharaceuticalcompositioncomprising anantibody toOPLwithorwithoutat

least one additional therapeutic agent, tobeemployedtherapeuticallywill

depend,for example,upon thetherapeuticcontext andobectives One sIled in

the art will appreciate that the appropriate dosagelevelsfortreatmentaccording

to certainerbodiments, willthus vary depending, in part,upon the molecule

delivered,theindicationfrwhichthe antibody to OPLwithor without atleast

one additional therapeutic agents being used, therouteof administration, and

the size (bodyweight, bodysurfaceororgan size)and/or condition (the age and

general health) of the patient. In certain embodiments, theclinician may tier the

dosage and modify the route of administration to obtainlthe optimal therapeutic effect, in certain embodiments, a typical dosagemayrangefrom aboutL 1 g/kg

T77 to up to about 100 mg/kg or more, depending on the factors mentioned above. In certain embodiments, the dosage may range from 0.1 pg/kg up to about 100 mg/kg; or 1pg/kg up to about 100 mg/kg; or 5 pg/kg up to about 100 mg/kg.

[0195] In certain embodiments, the frequency of dosing will take into account the pharmacokinetic parameters of the antibody to OPGL and/or any additional therapeutic agents in the formulation used. In certain embodiments, a clinician will administer the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages may be ascertained through use of appropriate dose response data.

[0196] In certain embodiments, the, route of administration of the pharmaceutical composition is in accord with known methods, e.g. through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.

097 In certainembodiments, the composition may be

admiisteredlocallyvia i antationof a membranepongeor another

aprata trial onto Wh he desed -olecule has been abooedor

encapsumte in eWain embodmerts, wherein mpan evice ,used, thedevicemay be mpanted intoany suitable tissue organ, anddever of the desiredmoecue may beviad sionmereasebolsoronnnous

administration

[0s iA cerain embodiens,iRmay be desirable tosea

pharmaceuticaicompositioncomprisg anntbad to PGL i th or withotat

leatoe additiocnalteapuiaent,inanex vvAnne.nsc itance, ces,t isues an/rorgans that have been reovedfr patientare

exposed to pharmacutica opposition comprising a ntiody to OPGL, ith

or withoutataone addionatherapeutagent Wh ithe Cils, tissues

and/ororgans areausuetym.planted backinto teatet

9I ncerta n embodment, an antibodytoPOL andAr any

additional therapeutic agents can be deiveredbyimpantingcertain celsthat

have been geneialy engineered, using methods suh asthose described

hereintoanepesand secrete thepypeptides ncertain embodimentssuch

cellsma,,,ybeanimalornum,,an cell, andmnay betoguheeogusr

xenogenic.Incertain embodimentshe elsm ybeimmalizednceain

ernbodinentsinordertodecreasethechance of animnol ogicaresponse,

the cels may be encapsuted to avoid filtratioosuroundingtissuesIn

certainembodiments,the encapsulatiomaterials aretypically bicompatible semi-permetbepolymeric enclosuresor membranesthat agow the release of the protnprodus Gbutprevent thedstuctionofthecells byth patient mmue system or by other detrimentafa actors from thesuroundingt ssues

EXAMPLES 020 The fong exampis, including theexpeiments

conducted and resusacheved are provided foriustrat purposes onyand

arnottowbe constuedasli.iinghe pree-nt nvnrton,

Cloning the OPG Heavy and gh Chains

[0201 HO celse pressing the engthhunan OPGL cDNA are

usedom irasD g ncicecotaininghumanim uno nenes

Lymph nodes from theimmrnized mice are fused tomurinemn acelsto

generate hybdomas.Supematantfromthehybridonalinesaretestedinan

E~iS~ssay OratboiSthat reactwithumanOPG ,LAIOOepes hybrdomnes AMG6mdAMG 64 and AMG are found to express

antibodiesw, ithhighafintforOPGL(Kds of0OZ28nMl:0 .8n.M:and0.23r.M3 respected yan rd AMG 6,5 selected frcloni n Heavyandlightchain ONA

Clones fomAMG65and AMG6 4areidentica and AMG6issed tocdone tOe aOght ChainA,w ie AMO6.4isused to dne theaCPGL

heavy chancNA.

$0

Ckonn fthe aOGL-1it chain

[0202] ThecaOPGL-1 kapahghtchain variaberegionis obtained

usingPCinRmplication methods from first strand cDNA prepared from AMG 6.5

total RNA. First strand DNAis prepared fromAMG &5 total RNA using a

random primer within extended 5'~adapter (S' GGCCGGATAGGCCTACNNNNNNT.3'(SEQ NO: 15))andthe materials and methods provided by theGibcSuperScript Preanplificaiion Systemfor First Strand DNA Synthesiskit(catno,18089-011Theo~gonucleotides

below aroused for the FOR:

$kappa RACE rimner:

- CATGAO CAGTOG;AAO AC CCT0 (SEQ D NO: 5) SapARACF primer

'.-AAGGG AG AGCA AAG GAT 3G (SEQ D NO:)

[02031 The ampfhed DNAsar clonednto pRTOPO

(Invitrogen arid theesultingpsmids are sequencedThe kappachain

consensus sequence is used todesign primersfoPCRampfcaonofhe

fui-ength aOPGLikappa chain.The 5'OPGL-1kappapimerincorporatesa

Xba site (TCAGA or coning and a OCACC Kozaksequencebeforethe

initiator Met codon.T- he3'QG-Kappaprmrncro ateSailsh, e ,C(GIOGA) fbfowingthgJestop cdon foconng

CAATCOCAG A 40 A AT GAA ACAGCG-' ,EO-NO;7)

Xb4Site Kozak I E P A (550DNOD 6) a"OOGL kappapimer: &-77ACOO IT,TATGACAO, TC dOCOCT GTT,-G-3, ($80 IONOCc5) Sal C G FZ N F (SEO1D NOl7)

[02-04 The fukengti aOPGLIkappc hbaineDNA clone is

obtainedusi the AMG5 3firsstrand cDNA descriedabove. byPC

amian with he 6 and3aOPGL-kappaprimers The POreaction

generates a 738 bpi agent encodingth2amerino addesdues(Mud

the 20 aminoacd :caninasqec.otea0PGL-1 kappan Chain (igure 4 8 D NO 4). Fowingpuri c using aQAquckPCR Puridfiction kit(Qaecat no28104,thifragment ',s usedtocnstucthe

kappalight chainexpressionvector.

[0205] The 738 bp fuiength kappafagmentgeneratedaboveis cut with land Sa spurred usingthePromega Wizad DNACleanup

System (Promeacat no A7100) and iclonedinto pDSR 9 to generate

plasmidaQPC tkappalpD$R 9 (Fgue 5). pDSRrhasbeen described

pieviously(seeVVO/ w4383w ihsherein ncorporaed byrerence forany

purpSoe e .gura e12)) eflomaepDRcdepDSR.2i{smodified

inthe fdowingwa the FSHpoiyA ishotened byapoxmately1400base

pairs,to 85 base'paks andnow ends thede te; thedihydrofolate

reductase ( FR) promoter ow contains209base parsa ngbeen

snortened from the 5-nd by approximately iloase;and anapproxmtely

basepairB§ ifragmentfomt e FR po yA sequence is delete,

The05G kappalighthbinexpressiodeneis sequencedtoconfirm that i oded fo the ame peptide this identified in the

AMO65 hybridoma.hein expkssnvector, aOPGLtkappapDSRrhEis

5476 bp and stains the 7functionalregions shownin abe2,

Table 2: Featres ofaOPGL-IkapppOSR1iS

!asmid Base Pa/rNumber 2 to 881 Atranscrptiontermitioion/poyadenylation signal from the acsubunit ofthe bovine pituitary glycoprotein hormone(FH) (GoodwinetatiNucleic Acidse. 1983 11: 6873-82; Genbank AccessionNumberX00004)

882to027 Amouse dihydrokate reductasa(DHFR)minigenecortaining the enogenousmouseDHFRpromoter,the cDNA coding sequences, and the DHFR transorption termination/poiyadenylaonsignals (Gasser et a lProc NAAcad SUS A 198279:6522-6. Nunberg et a Ceo!1980 19:35564 Setzer at a J BA Chem, 1982 257: 5143-7; MGrogan et , J BlotCham 1985 260: 2307-14)

2031to pBR322 sequencescontaining theampicinresistancemarker 3947 gene and theoriginforreplication of the plasmid inE col (Genbank AccessionNumberJ1740)

3949 to An SV40 early promoter,enhancer andoriginofrepcation 4202 (Takebe at Mo CellBlot1988 8:466-72., Genbank Accession Number J02400) 4299to A translational enhancer element from the H TLV-i TRdmaln 4565 (Seiki t al, Pm Nail Aced Sc U S A, 1983 80: 3618-22 Genbank Accession Number JD2029)

4574 to An intronfromthe SV40 168, 18 splice dono/acceptor signals 4730 (Qkayama and BergMo CellBlot19833:280-9.Genbank Accession NuberJ02400)

4750 to beaOPGLKappa eight aia n cNAbetweentheXbaandSal 5476 sites

[0207 A crcar plasmid map of thevectris showningure5.

Clornng of the OPOL1 heavychain

j0208} TheaPGL-11kg0'2hevobiisclo10ned rnAMG 6A

hybodoma doube-strandedoDNAproduced withthe Clontech Maathon cDNA Anm anKit(cat. na om802- An acof A eavyc oNA is scompishedby 5 and rapidamplification of DNA eds RACE)

echniques perfvedwh human gemine G2heavychain.onstantregion specificprmers (shown beow) andRACEprmersandothermateatsand

methods povidedintheMarathonVeDNA amploation kt

&'g02RAGaprimer

'GGC AGOGTC0ACCOAGO0TG0TGAG4 SE10 NO.;9) 'IG2 RACE pimer 6- OCTGA OQAAG GCC CGGGTOT- S 10 O 10)

0209 The 600 bp 5 RACE rodut and 1200 bp RA p duct

iNOrumin isused to designQr,,CPGL-1'he a-vyc 'hainspecifiprimersto the ckning otfullA -lengthseuec.hehevyhin: ie(5oOP k-IGQ Prinerisirted againstteenestand andha a s Hin1siteand

consensusKozaksequencebefore the naturalstar site, Te heavy chain

primer (3aOPGL-I gG2 Presanbaisense prmerhatconaina Sagste

a'stop:odonaterthelast amino acid of theheavchain@02sequence

(SEQ) 10NO; M 1$

3? 2 ]OPOLgI N2 A erimern

G KSP L 3 (SEQI0 NO n19)

[02101 The d eoDNAdcbaboDaveisusedpto generatethefu-egt heavychiGn DNAebyPa atinwithe5'-A pd

3-acPGL afy0pAimersT etgenertesaa1433bpagmen tencong

thie4i7 am--nc,id resies'ncluinghe 19 amino acid IgG signal sequel ance) wofmaecxOPGLlgG2 heay chin proein(FigurE,.ID NO; 2.Folio:> ing puifciinuigafquikPO rfctin Qaecatn 14)ti fragment Isusedto constuct the heavychAinexpeSSonvecorasoslows,

[021 1] DN AeDn codi.ngtfl. ieng th g2havyfagmet

gene-rat ed above'isc utwi"th idiand aipurifled usingaf qukkl Exrct-ionit(agencat,no&8704),andthisfragmedtIcloedi01tODS 9,~

Theesutigexrsopiasrilidis namedPL1IGJD cd(iue)

A;ecocopoetsridentcaltoaOG-epap$c9vetr

aOPGL-1 kappalightchain 0NA between thetaand SM sites,TIhe tOPGL-

I IgG2 heavy chain expression clone is sequenced to confirm that it coded for

the same polypeptide that is identified in the AMG 6.4hybridoma,

Example 2

aCPGL-1 expression in CHO cells (0212 Stable expression of aOPGL-1 antibody is achieved by co

transfection of aOPGL-i-kappa/pDSRaI9 and aOPGL-1 -IgG2/pDSRu.19

plasmids into dihydrofolate reductase deficient (DHFR-) Chinese hamster ovary

cells (CHO AM-1ID, U.S. Patent No. 6,210,924) followed by isolation and testing

of individual clones.

[0213 A 100 rnm tissue culture dish is plated with 1.5x106 AM-1/D

cells grown in CHO d- medium (DMEM-high glucose, 10% fetal bovine serum,

1% penicillin/ streptomycin/glutamine, 1X NaPyruvate, 1% nonessential amino

acids (NEAA))(Gibco) and 1% ht supplement (Gibco@)) the day before

transfections (Day 0), On day one, 400 pi of serum-free RPMI 1640 medium

(Giboo@) is aliquoted into a 12x75 mm polypropylene tube. Twenty four microliters of TransIT-LTI reagent (Mirus Corporation) is added dropwise to the

medium and the mixture is allowed to incubate at room temperature for 10

minutes. A total of 15 pg of linearized plasmid DNA (7.5 pg of aOPGL

1-kappa/pDSRa19 and 7.5 pg of aOPGL- -lgG2/pDSRai9, digested with Pvui)

is then added dropwise to the mixture and is incubated at room temperature for

minutes.

10214] The CHO d- medium is removed from the cells, which are

washed with 10 ml of Dulbecco's phosphate buffered saline (Gibco@). Six miliitesoferu-freM ~medusupplemerited withTL-gk±,NEAA and Na pyruvate (Oibo) s added t the cellsTheDNtcomplexisadded dropwise to the plates, which are genty rocked back and forthtodistutethe

DNA evenlyoverthe cilAfter 6 hous inatissue ulturencubato e

medium isreplaced wh resh CHO-medium Forty eighhours aterthe ees

are split toted 100mmoure'dshesnUCelctmdim(D E i-gh gucose,10% dialyzedfetalbv ine seum(BS 1%pecnstretomyoin

/gutane,1%nonssenrai noacids and XNa pymvate)ib®@

Medmischangedteek ti coodies appeared.

[02151 Afer 1014daysclonies re piked using 5 mmc onng

diss (Labeore) soakedin 1x-ypsin DTA(Gibo) and ar cultued i24

welltissue cuture plates withCHOselectmedium.When the ceis become cofuent,serumfreemedia (QO1 select medium mmus FBS) added and is

thnclected 48 hours later These conditioned media areanayzdfo

blotwhhose eoxidaseHP)

conjugated goat nthuman IgG Foanbody (PierceRookford L to detect the

OPOLI heavy chain,and goatant umankappa chainantibod (Pere,

Rockordd ifol owedby HRP-conjugated abbt anti-goat gG(H+L)anibody

(PfrceRoeordILto detectthemiORAGL-IlighchaW ih, ehihsexpressin-g

cones arexpanded and stored in qud nitrogen

Excam pie ProductionofPGLni Preparaton and Creation ofCell Lne '2r0

[02161 CHOSelsproducng 0PLiareclned by ,wrouo citing dlonin 90 welp tesndersemrfreeconditns.Theclonesare

selected based onproduconandgrowh chatesticsnnaous suspension

vessels.EIAs aeperdormedtoselect heclonethat produces the ighesteve

ofcORG, -trowthhrcestc nk dindoublingtimlresnd detn-sitiesa re then measured by growing the ineen 100 m 250 ml500 l,-L:ad3L spinn~er fla'sks:as wellasin$LAplikon bioreactors. TheclonewhhITthe fastes-at doublg i me thatreac es theghestdene tyincultureisseected andis

designated elline1250.Whenteleaspndtoidufcet c ellstofrez350ampulesatppoimteyxi elsrnLcels are resuspended n aryopresave, femed (%tSoy Mediumf(seTablefodet .ails)supplemented with 10DmInonessentiamno acidsanmd I0 miL&Gusnn(iclLilvtoe)and 10%dirnethy -sulfoxie(JT ae))nfrozen. Ampulesaare store d in aiieacsfaclliy and aresubmegedilqudaroage iniquid ntrgendar.

10217] Based on rowth and produ in Smanwscaeainners and langerscalebior co s ,Ceaine1250.schosenasthecellinfor

rnanufacrturing cfoQPGL-t

Sa

[0218] a- oGL-1 is produced by expressioninCell Line 1250,

clonalline of CHO cels that expressesaOPGL-fromplasmidsaPGL

1-kappalpDSRa19 and aOPGL- QgG2pDSRa9. Thcellcultureprocessfor

OPG- iisshownin Figure 19. For each produoIon run,cells from ial ofCell

Line 1250 are iniilygrown in $0 mL of VM-Soy Atch Medium (see T sae 3 for composition)supplementedwith 10 mlL nonessentialaminoacidsand 10 m/L Lglutamine (GibcoLTO/nvitrogen)(VMSoySupp) in 125mi erlenmeyer shakers

at 100 pm for 5daysThe entireculture is thenusedtoinoculateVMSoySupp

a500 ml spinnerflaskto 3x10viabl ces/mi (3E5vm ), andisgrown with

70 rpm spinning for 3-4 days The entire culturefromthe 500 m spinner flask is

then used to inoculate VM-Soy Supp in a L spinnerflask to 3E v/mi,and is

grown with 70 rpm spinning for 3-4 days.

L02191 The culture from th 3 pn fakthpinnerlaskihen split to two 3 L spinner flasks at3ES v/ri in VM-Soy Supp without phenolred and grown under

the same conditions, These spinner flaskculturesarethenusedtoinoculatefour

additional spinner flasks at ES vC/m in V-SoySupp withoutphenol red, and

grownunder the same conditions, Four terms of culture fromthe our 3L spinner

flasks is used toinoulate10LofVM-SoySuppwithoutphenolred in a 20 L

bioreactor, andthe bioreactor isrun in fed-batch mode for 7-10 days, in

fed-batch mode, a nutrient feed containing concentratedmediacomponents

("eed" assetforth below in Table 3) is added to maintain cell growth and

culture viability, f0220] Thedie trcuieeom the 20L bireacois thenusedto noclate 70 o V Soy Supp whout phenol rediiara0lLbioreatorand the bioreactor in fed-batch defor 9-1day Finaly the entire cutre rom t-het5Dbiomaotor is used tooculatappxmAaeyO LofVM-3Soywiht supplementor phenolredin a200Lbioreatorand the bioreactoris mnf ed Etch mode, The rate offeed durngfed-batch nde ia determined such thathe gAuCoseleVe ithe Culure a taInedat 06 ore ioreactor cef density and ucose concentraton are measured di andthe rate of feed adjuste;-d'accodingly

[021 Production in th 2000Lboreactorlasts foapproxmatey

two du ringwichtinae ris ti producedodbythece uad secreteed intothe celcuture -medium.

[02221 The production reactor ico trolled at set pH, temperate and dissoledoxygen level- rpHisT,7,),and iscoontIled by carbondioxide gas and sodim cro e Adodissov oxyRgen is120ini~gadisonroledby

airknitrogen, and, oxygeng-asflos aremantaiedat,7Cthroughoutthe procesA ges are passed toughmembrane tersof poresze0 22pmor

j022] Attheend of production thecellbrothi sfed ,itoadis, stack centrfugendheulurspemr.atantissepar-ated frmthe ceals- Thentrt

is frtherc through aCuno OSP depth rfollwedby a 2p Posidyne fitcer(PailCo, ThelArificondtonedmediaisthen concenraed by tangentiallyo trailtrti(U)uig nSOkD N L membranes(Millipor

Thmoax 50) a T oriionedediaisconcentrated1so3&-fdhihe

resultngconcentrated conditioned medium (0CM~ishen either processed through puriicatinor rozen for purfcationatatedatehe producio

processssummariedin Figurei9

CellCuue Medium t0224] The celcultue medium fr usehoughoutthe enecell cum reprocess is basedon Dulbeccos ModiedEage Mediumtam u t F12 (iMM/F12,t and contains supplementaeves of amino acids, addition nutriensand salsa soy hydrolysatean combnanthuaninsun

(Nucelln ZnEIiU LiyThe omponents areited in Table3 Imedais referred to asV Soy. Mediasoluodnsarefiteredtroughmembrane fmersof

02 pmrpoasize prior to use,

Table 3. Ce Culture Media Components

COMPONENTS FOR BASAL Media and FEEDS_ _

COMPONENT VM BthMed i( LE (n/L)l

DMEM/V12COMPONENTS -------- Inorganic salts CaC (anbyd,) 116.60 233.2 CuS045HcO 2.0026 0.0052 Fe(NO3)39R2'~0 21000 0.2 FeSO4,7H20 O,8340 1I6

MgcO*(anhyd.) 57,280 114.56 MgSO4 (enhydd 9768 1 i95.36 NaCl N181L98

Na1T2P.H20 125,00 DO a2PO 42.040 284138 ZnSO47R2 0,8640 *328

Other Components D-lucose 3151.0 12302 Na Hypoxanthine 5,40 i0 Linoleic acid 0,090 0.18 Lipoicacid 0,2060 GA12 Phenol Red 810 162 Putescine 2H 01620 0324 SodiumPyrnvate 110.00 220

Aminoacds L-Adanine 26.70 53A L~Arginine H~l 295.00 590 L-Asparaginelk2Q 45,00 90 L-Aspatcacid [39,90 79,8 L- Cysteine-LH-2 35.120 70.24 L--Cystine,2HC1 62.580 125.16 L-Gumac44.10 88.2 L-Giutai 657.00 1314 int 52.509105 L-Ristidine.HCLH2O 62950 12i.9 L-Isoleucin1e N217.88 L-eucine 118.10 2362 L-LysineHC] 182.50 365 L-Mthionn& 34.480 68,96 L-Phyanine 70.960 14192 LProline 57,50 115 L-Serie 73,50 147 L-Threooine 106.90 213,8 L-Typtophan 18 36,A8 L-yrosine.2Na2Hi20 11,.580 1 223.16 L-Valine 10570 214

vitamins Biotin 00073 0,0146

D-Ca Pntohenat 4,480 8.96 Choline Chloride J17 960 35.92 FojicAcid 530 10,6 l14noSitol 25.20 50.4 Niacinamnide 4,040 8.03 PyridoxaiTJCI 4,00 8 PyridoxineoHCI 0,0620 0,124 Ribojavia 0,4380 0.876 ThiamineBHC 42340 8,68 Thymnidine 0.3635 0.727 Vitamin B12 1260 2.72

AD)DITIONALCOMPWONrNTS 1 _ _

Nucellin Zn,(rhuiinslin) 500 '5 Selenous Acid 0,0050 0,015 Triiodothyronine 0,000040 0,00012 Hydrocortisone 0,020 0,06 Ferric Cifrate 1122,450 122,450 Piuronic F~68 1000.00 500 Soy Hydrolysate 6000.00 6000.00 NaHCC3 3000.00 3000.00 NaGI 3500,00

Pridfication Proces

p25J aOPGC~TexpressedinO deilsisecretedinto the extacelurmeiumA seeaofsteps maybe usedto generatepur ematerial

The process uses hydropnobicchargeindutoncationexchange, and hydrophobicineractiontchromiatography alonowthaowypH step andiraffilte 4 Theseproceduresaredescied bIow

AHydrophoibChards ndciZonChromatography $ C

02261 This hrometography step removes hemajohityofhostcell

proteinsanL`NA The concenated condoned mediaCCM isfiltered

;rou aCuo $Piterab enoug a CnoROmchred celllosebasedfilter,and thenloaded on to anMEP HypCeresi, After

aadn the coiun iswashed with equibration buffe(20 i apH 7) he anybody is euted fromtheresin using alowpHbuffer(20 MSodiumAcetate

pH 5 A0) its e ted from thecolumntheproduct is oected baseon the absorbance at 280 nm ofthe column effluent

0227 The EPpoo is titratedto pH 3and held for aproimtey5mnteto inacti'vatePot nillynamiatngerirs Followingthe od step episadustedt approximately

C Nrailration 0228 The pFbajusted podis fleedthrou a ipeVre esove

NFR fitr or equiaentThe antibodyflwsthroughtheflterwhilepotentialy

contamiatngvirudson50nrm areretained.

.Caton ExchangeChromatography(CEX)

f029 The antibody is further purified bycatonachange

chromatography usn SP Sepharose HP Amerham Pharm..cino equivalent The cation exchange chromatography stepremo'es additonal0Mcel

proteinsDNAlowermoleclar weight proteins, and aggregated forms of OPC'. Theviral filteredpoolisloadedonto thecatoexchangeresinAfter loadig,the column is washed with equlibrationbuffer(20mM NaMES pH 2), The antibody is then ed with alinear gradientof increasing salt(20 mM

NaMESpH,2,0 MNaCi to0 mM NaMES pH ,2 0.3 MNaC, Asit is eluted

fromthecolumnthe product is collected based on the absorbanceat 20 nm of

the column ffluent

5HdropoobieracionCromatorapy1 a0 02301 Theantibody is furtherputed by ydophoiintecon chromatography using Pher ,yToyopead 650$(TohTiosept oreulen The

hydrophobic neratonehrnratogaphy step isUsed assatpeng p and reovesadd.tisooNa i0 ellprotein NAom leculareightproteins and agrgtdomofwOPG&1. Thecat.ionexhneolsodtoebefore

loadingonto thecolmby addtionoNf ammonium sulfate to aconductMtof 105mS/cm atI15=50.Afterloading, the coinisw lashed with the

equilbratin buffer(1M otassium Phosphate ph 8) Teantibodystheneuted

with a inar gadient ofdecreasingsalt concenratin (1MPotassium hosphate PmMTrH 'S to OM Potassium Phosphate,20 mM Tris pH 8)As is eis ted

from the column the productisoletedasedon th aso28ance at2omor

thecnoumn efen. Concentaionand Dafiltration

f0231 he HIC cumn poolisconcentrated anddafleredinto

formulation buffer by tangential fow ultrafltronusing 50kD NMWemembranes

(Miporeiomax50).he formu aCn uffrine 10mMAcetate, 5 Sorbitol pH 5.2 and iOPGis at 30 mgimL ila Flratonand ae

[0232 The purified bulkisassed through 22rmFPVDF fiter

(Miiporis samped, andstored at approimately3C ina securedfeezer,

Example BindingSpecificityof aOPGL& (0233j Antibodiesthat are oduce CHO fellsthat are ransfeted wth the twoexpression vectors as discussed n Examples I and 2

may be Med Aithe flowing examples 4 5and 6.

C02341 Human OP binds and neutrazes OPGL in smiceand cynomoigusmonkeys, as we as n humansa.PG1 binds urmanCPGL With

Ih affniybutdoes not inds gnificantlyto mudneOPGL (Tbe4

Table 4: Affinity of aOPGLA to Cel Membrane Expressed OPGL ofHuman CynomolgnasMonkey, or Mouse Sequence,

OPGLSpecies aCOGL1A ------------- E- (n- -i Uumnan 16 Cynomiolgus 19 Mouse No Specific Binding OPGL of thesespecies is expressed in CHOcesasthefl-ength, membrane-bound protein Binding of coGL to the esurfaceexpressed OPGL is assessedby FACS analysisofcls incubatedwithOPGL and ai-ibledsecondary antibodyto humanlgG2, aPGt-1 binds human and cynonolgus OPLbut thereisnospecific binding to mouse OPGL

Q235 Inadditon, human OPG has been report to showweak iding to igandTR (morTneosisataor;ead roe eataW 2000 related member of the TNFfaUy, whichshows DNA ad amino aidsequencehomology toOPGL Lacey et aL,9M), However:OPS does no detatabbd to otherTFEated proteins such asTFaN or

-23C1 cOPGL bh n specifical toPOL on pates (igure 7)Recombinant souble OPL (2 pg/m)is coated onto96welIA pates at

room temperature for 16 to 24 house, Ater blockigwith 1% BA in PBS

varyingconcentrations ofo CPQG appoximty2np/nito 1000 ng/midited

*A BS A/PnS ae added to thewellsadtheplatesare nubnated forabu2, hours atrom temperature Bound antibodyis.detected oatantiHuman

gG (FaelORP using TMBH202 (tatramethyhenzdinead hydrogenerxde)

substate cocktail The absoibanceisrad atOm and60m

j027 oPGL binds spe ctailyOPGLexpressed onthe surface ofransfe-cted cells (Fire ,rXn8)OwPGLI1(10np/m)diluediFACS" uffer (PBS,0Mi%BSA,00%o u~ieispriobaawtvaying cocetrtinofOPLNFa,T-,NFt.TRAL, or0CD0ignd(appovimate'y 0". np/~t 00n/miadithnaded to appoxima~tely 20000CR0RENI 218-9ceWln,whichare CHOcetsAblY expessigebneoidPO n thec reNlsure After 1hour ata2n8Cmnoundanbody Iecaed by

cantrfugatonad wash ng. Ces are then ncubatedfo 30mnutesat 2-C

we F(ab) 2 GoataniRuman 1§3 (cyfragmentapecc)A Aer cenrtrifu atondwas ' zc lsurfacn f essence IsmesrediOng ow yometry gure s8shows thatbinding of oCPGL1to CHOREN29`cell is

:17 specific, and is copetitivelyreduced by addition of soluble OPG 4 but not by addition of TNFa, TNFTRAiL or C0D40 liand,

[0238} In competition experiments, OPLbinding toOPGL or ERA plates is inhibited by addition of exogenousOPG,, (Figure9), but not by the

addition of TNFz TNFP TRAIL orCD40ligand Figure0 This procedure is

perfomedin substantial the sameanner as aboveFo binding of tOPGto OPLon E1A pates except acost oncenratonof cOPG-i Ong/nmLIs

preincubated with varying concentrations ofsolubleOPGL orotherliands approximatet yIng/nit'O000 ng/mifor each) before added to the OPOL. coated plates,

Example 5

aOPGLiNeutraingAciity Inhibition ofOsteoclast Formation

(0239] RAW 2647 (ATOCMoT IB7,Maassas A a amune nacrophage celllne that was derivedKom an Abeisonmunnleukemiavirus

inducedtumor RAW 264J ellswldifferentiatetoosteoclastCikecels inthe

presence of OPGL. The basic assay forgeneration fsteolasts in culture from

RAW ces in the presence ofOPGL has beedesbedindetain Simonet -ta 997) Ce//B0p 309, and Laceyet al (1998) Cellp4 5, which areheei

.orported byrjeferencefor any purpose.

[0240 RAW cellsarestimulated by ligand to differentiate into

osteoclast-like cells, and the differentiationcan be measuredby TRAP activity, a pronerty01OsteoadastsThus the effectaOPGL- onosteoclasgenesisa n be measured.

024 RAW cels are noubated fo 4ay inthe presenceoa constant amount ofOPGL (40ng/m n6amountsofPG Dndvy (63 ngito 200 n/m in cedcturemedium (DMEM' F0Sj ,292 mg/mL Gut 100 urts/mi PeniinG 100 pg/ teptomycin su ate tthendof4 days, the elsarestainedfor tartreereistant acidphosphatase(APac permea atandacnd caonfo owed bytreamenth psamnitrhenyp hosphtfor iutsBieflythmediasasiaeoffof th'ec, ls-'a..d1 00 plofitrate buffer (410mrlDA citrcacid.5gmi0,1Nm citratetriodi usat, n tiron 4 CC)isadded toeacwed andtheQlasare inubated for 3to 5inutesa room temperaturOne hundredicroAtesOf P okuiton i en added (6 mg acid phosphatase rage (igma04 00)7.2 m ouiIon Sate (Sigmacat n.$$ and 218 ctrate aendplteareincub-ate-d for -to 5minutesat romempretreTherePactio)nis term d by ad on of50p05M'e" soltion.

R2,12 TRAwillconvert par-nirphnlopa atooa nitropheno which can be uantiatedby opticaldensy measurement at405rm TeTRAP actcaa urrgae maerfor osteolase pme thereforecorrelatesMt the opticaldensyat 405nA pt ot opicadensty vemus OPGW ionisshow centaton inFigue 1,and demonstrates that IOPGInhibitsoasteockst formaiioninthis assay jnhlbftiorDfCPGLinding to fs Receptr

[0243] The potency of uOPGL- is demonstrated by its abiiy to

block the binding of OPG, Ogand to its cognateireceptortheqteoclast

differenatioand tivatngreceptor (ODAR, also known as RANK). This assay uses homogeneous time resolvedfluorescentresonance($TRF)todetect

binding of aOPGL-1 toEuropiumconjugatedosteoprotegerinigand(Eu-OPGL)

if OPGL-inhibits Eu-PGLbinding to ODAR, fluorescentoutput widecrease,

and the amount of OPGL present wi beinversely relatedto the amount of

fluorescence.

044 OPGis labeled with europium, which emits light at 620 nm when excited with337 might< ODAR is fused to FLAG and to Fo, andtheF

ODAR-FLAGfusionprotein is absadwithananti-FLAG antibody 0nkedto

aophycocyanin(ACafluorophorewhichemits665 nn lightwhen excited by light at 620 nm. Therefore, when Eudabeled OGigandbinds to the F-CDAR

FLAG/anti-LAG-ACcomlexthe tertiary complex wi emit 65 nmight when

excited withlight at 337 nm.

[0245i Eu-OPGL at 00 pg/l is preincubated withvarious concentrations (1 to 150 ng/i)of aOPGL-1 in assaybuffer (50mM Tris pH,

100 mMNACL ,05% NaN, 0% A, and 005%Tween 20) atroom

temperature for approximatelyone hour (Preincubation mix), A mixture of Fe

OJDAR-FLAG (I pg/m) and ti-FLAGAPC (2,5 pg/mi) isalso prepared in assay

bufferand incubated at room temperaturefor one hour(Fluorochromemix

Equal volumes of Preincubation mixand Fiuorochromemixare thencombined andincubatedarontemaperatureor hours. Theuesene byaiplaes one ackard DccveryrHT P mFropeiateanaly7er excitation waveljengthof 337 in-iaanemssonavlegtof6$nS

[024S) When aOPG sL-1reincubated wtuQPigandandis

then mixed with a a ohe o enes at 665inndecrease in a dose dependentmanner asshowninFigure12

demostraingtatuOPGLI62 can efetielinhibiOFG. bdingtoQAAR

Example S

Ptarmacokinetics in Cynomolgus onkeys

f02471 S male and sx female cynomogus monkeys, not greater

than4 5 years of age and Wehing 2 to 4 kg are assigned to 4 dose groups Group consists of males and ferna es Groups ,arnd 4eahconsistsof 1 male and feame An main Group ae ;adminsteedasingleS doeof

mg/kg OPGL- ,hleanma sn Groups 2,3 and 4 areadisteredsngle i dosesof0 1,a ,r10mgkgofresgectively.

[0248 Anmalare dosedwith OPGL expressedfrom transfectedinesehamsteovary(CRM clHO Srusaplsa eknr

eermnatinof oCPGL-1leve6,antibdy anali,an-danlyisfheb',one r rn ea er teopept8de(ser.umn-Tx),ka~ephosphatase ,,AlP

and se calcium serum0 Urne islsocleted for analyss of

e ropeptidurn eNrTx)erand $reatinQe

[02491 The serum cncentaion--iaeprofiles.folowing 1V administraton are characterzedbDyarkphakcisrbuin(iur1)Initiplly

if there is a rapid distibution phase,foowednyasinificantlyslowerplateau phase, which appearsto be concentration-dependent The third observedphase is arapi eimination phase t02501 Nonompartmental analysigof complete serum concentrationtime profiles using WinNonProfessonalvI.5)andexponentia analsof the data up to 14 daysafter test arceadminstration and above

10000ng/msngA SAM H (v1l2) are utilized oinvestigatethe pharmacoineticsdofoOPGL in monkey The itia volumeofdisibutIonfm

all doses averages, m k , similar to plasmavomeSteady state voume

(V/') of distribuon averages N mikgacrossal 1VdosesEponentanalysis

indicates that aPGL1 has an average distribution ha life (t iof 02hours, an extendedsecondaryphasewith a haflife t that increases withdose from

86,9 hours at dose of0,1 mg/kgtoamaximum of 444 hours at adoseof 10.0

mg/kg. Terminal eliminationbhfe(tZ)estimatednon-compartentaly

averages 31 hours across all N dose groups. Clearance(CL, CLF) of aoCPG

is found to be non-iinear, with animalsreceiving dosesof 10 mg/kghaving an

average clearance (0120 milhr/kg) that is 3,3fodlower thanthose receiving 01

mg/kg (0.401mlhr/kg).

(0251 After administering subcutaneousyabsorption is slow, with

ave agepeak concentration (Cr )of 11600 ng/ra132 hrThere ishigh

variablty in the range of exposure fter SCa dministradin resting nan

average clearance of 0387 t0128 Ihkg andrimeanr idenetie of 202 801 hours Averagebiavaiabiltyis89%

2$ ThepresdfngdaaresmmrariedinTble5

Table :Mean( SE NonGompartmentaliPharmaokinet Parametersin CynLus Monkeys Administerin an Do f Q0PGL- IV and SC Non-Cempartmiental Paramneter Estimates ___mg/kg___0_ mg/in~kg LOwmglkg i mg/kg Pmtr Unit SC (n=6) IV (riF2) IV (iF2) IV (nn2

Tramehe 132 612 ?A0a 0vt Mea

Cep hr IRg/nI 11600 34,9 f11d 3410 43310 302 38200 31A.ND 3200

AUy ~tghr/i 3520 1730 253 3950 9900 CL, CL/F mrnkcg 0.387 0.281 0A01 0256 0.120 MRT b 202 803i 84.8 124 1 519

vamd/kg N NA 33 ~/ 31.7 SS 9 t Dd~aermnd, PK Mnpies eMdddgpaa 0 hs eme~nlcaei o bue *Not AppUmde

f0253 oOPGL~causea rpid decrease eiemfT xleve within24 hourspostdose(Fiure414).The average time of mairumreffectis 10...........23-------- observedtooccur between 12hours and 7days post dose as Vdoses increase

from0GA1to 10mg/kg and betveen1hours and11daysnanimasrfecevna SC dose of.0 mgkMaxmumrreffect increaseswith doserom appoxmaey

furthersup ressicnis observed wth maximuminhibtonof i% Mean levso f serumhbNxreturnto baselineby day28ateradministeringG0 mg/kgad by day 70 afteradinisterin g/gkg SC.UrineN-Txshows similartrendstothose

ofseumNx~exepthatalgrUpsreturntobas$e nevalues by studyday1$05 (Figure15)

[0254] SiuppressionofseruCaincreasesvifh dose toa-3mjean ni rof 311% WowthebM aneveragesevedays adm trtonof '0 gAother dosegroupshavemeandCaofess than 264%fRomthI&rbasie averages.SEy day 17alsuCalevelsi!n trated animalsrtur to ithin10of therbasene averages Fgure 20.

[025M Asboneresorptl andformaioareinimatelyike

changes in bone fora inmarkers (ALP)are ao observed wih amuchowr decline inALP sand a more pronged spressiontharhe fomain

markerNrx(F igu r2Observig one esorpionmarkers decree porto

bone format markers (ALP) flowing dosing withJOPGL1 confmsthatthe

ciOPOLlis a bneanthresorpiv-e aganL

[0256} The aor of anim ls(9 of 12) developantibodies to

aOPGL-1, The ncidenceof antibodiestoOPGLisndose route

dependent itisnopssibleto assess the ef etof anibdies toCPGLon

ntboyneat and posie Ianma l 0mgg the ajMrit ofmOPGL 1iscla edporto anoydeve opmenanderore effees CL dispositon ar not observed(Figure16

Claims (32)

Claims

1. An antibody or binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises CDR1, CDR2 and CDR3 of SEQ ID NO: 13 and wherein the light chain comprises an amino acid sequence of SEQ ID NO: 14 wherein the antibody interacts with an osteoprotegerin ligand (OPGL).

2. The antibody or binding fragment thereof of claim 1, wherein the heavy chain comprises a first variable region comprising the amino acid sequence of SEQ ID NO: 13 and wherein the light chain comprises a second variable region comprising the amino acid sequence of SEQ ID NO: 14.

3. The antibody or binding fragment thereof of claim 1 or 2 wherein the heavy chain comprises the variable region and the constant region of SEQ ID NO: 2; and the light chain comprises the variable region and the constant region of SEQ ID NO: 4.

4. The antibody or binding fragment thereof of any one of claims 1 to 3 wherein the antibody comprises

(i) a human IgG2 heavy chain comprising the amino acid sequence of SEQ ID NO:13, and

(ii) a human kappa light chain comprising the amino acid sequence of SEQ ID NO:14.

5. The antibody or binding fragment thereof of any one of claims 1 to 4, wherein the antibody is fully human antibody.

6. The antibody or binding fragment thereof of any of one of claims 1 to 5, wherein the antibody inhibits binding of osteoprotegerin ligand (OPGL) to an osteoclast differentiation activation receptor (ODAR).

7. The antibody or binding fragment thereof of any one of claims 1 to 6, wherein the heavy chain and the light chain form a single-chain antibody.

I-1C_

8. The antibody or binding fragment thereof of any one claims 1 to 7, wherein the heavy chain and the light chain form a single-chain Fv antibody.

9. The antibody or binding fragment thereof of any one of claims 1 to 6, which is a Fab antibody, a Fab' antibody or a (Fab') 2 antibody.

10. A pharmaceutical composition comprising an antibody or binding fragment thereof of any one of claims 1 to 9 and a pharmaceutically acceptable excipient, diluent or carrier.

11. An isolated composition comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a heavy chain and the second polynucleotide encodes a light chain, and wherein the first and second polynucleotides together encode an antibody or binding fragment thereof of any one of claims 1 to 9.

12. The composition of claim 11, wherein the first and second polynucleotides are part of separate nucleic acid molecules.

13. The composition of claim 11 or 12, wherein the first polynucleotide is part of a first vector and the second polynucleotide is part of a second vector.

14. The composition of claim 13, wherein the vector is a viral vector.

15. The composition of claim 13, wherein at least one of the first vector and the second vector is a viral vector.

16. A method of treating bone loss in a subject comprising administering an antibody or binding fragment thereof of any one of claims 1 to 9 or the pharmaceutical composition of claim 10 or the composition of any one of claims 11 to 15.

17. A method of claim 16 wherein the bone loss is associated with at least one condition selected from osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, osteopenia, osteonecrosis, an inflammatory condition, an autoimmune condition, rheumatoid arthritis, and cancer.

18. The method of claim 16 or 17 wherein the subject is a human.

19. The method of claim 16 or 17 wherein the bone loss is associated with an inflammatory condition, and wherein the method further comprises administering at least one additional therapeutic agent selected from an interleukin-1 (IL-1) inhibitor, IL-1ra, anakinra, a TNFa inhibitor, a soluble TNFa receptor, etanercept, an anti-TNFa antibody, infliximab, a D2E7 antibody, a non-steroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, celecoxib, rofecoxib, and leflunomide.

20. The method of claim 16 or 17, wherein the bone loss is associated with an autoimmune condition, and wherein the method further comprises administering at least one additional therapeutic agent selected from an interleukin-1 (IL-1) inhibitor, IL1 ra, anakinra, a TNFa inhibitor, a soluble TNFa receptor, etanercept, an anti-TNFa antibody, infliximab, a D2E7 antibody, methotrexate, a soluble form of CTLA4, and a modulator of glucocorticoid receptor.

21. The method of claim 16 or 17, wherein the bone loss is associated with rheumatoid arthritis, and wherein the method further comprises administering at least one additional therapeutic agent selected from an interleukin-1 (IL-1) inhibitor, IL-1ra, anakinra, a TNFa inhibitor, a soluble TNFa receptor, etanercept, an anti-TNFa antibody, infliximab, a D2E7 antibody, a non-steroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, celecoxib, rofecoxib, leflunomide, methotrexate, a soluble form of CTLA4, and a modulator of glucocorticoid receptor.

22. The method of claim 16 or 17, wherein the bone loss is associated with cancer, and wherein the method further comprises administering at least one additional therapeutic agent selected from a keratinocyte growth factor (KGF), a KGFrelated molecule, a KGF modulator, an anti-Her2 antibody, an anti-CDC20 antibody, and an anti-EGFR antibody, and a PAF antagonist.

23. The method of claim 16 or 17, wherein the bone loss is associated with cancer, and wherein the method further comprises administering at least one of radiation therapy and chemotherapy.

24. The method of claim 23, wherein the cancer is selected from breast, prostate, thyroid, kidney, lung, esophageal, rectal, bladder, cervical, ovarian, liver, gastrointestinal, multiple myeloma, lymphoma and Hodgkin's Disease.

25. A host cell comprising the composition of any one of claims 11 to 15.

26. The host cell of claim 25, which is a prokaryotic host cell or a eukaryotic host cell.

27. The host cell of claim 25, which is a mammalian host cell.

28. The host cell of claim 27, wherein the host cell is selected from a Chinese hamster ovary cell, a HeLa cell, a baby hamster kidney cell, a monkey kidney cell, and a human hepatocellular carcinoma cell.

29. A method of producing an antibody or binding fragment thereof that interacts with osteoprotegerin ligand (OPGL) comprising culturing a host cell of any one of claims 25 to 28.

30. An antibody or binding fragment thereof when produced by the method of claim 28.

31. Use of an antibody or binding fragment thereof of any one of claims 1 to 9 or a composition of any one of claims 11 to 15 in the preparation of a medicament for treating bone loss in a subject.

32. The use of claim 31 wherein the subject is a human.