AU9134198A - T-cell antigens, and their use in diagnosis and treatment of T-cell mediated conditions I - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION .h NAME OF APPLICANT(S): to** Andrew Dale Weinberg AND Arthur Allen Vandenbark

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ADDRESS FOR SERVICE: S* DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street Melbourne, 3000.

INVENTION TITLE: T-cell antigens, and their use in diagnosis and treatment of T-cell mediated conditions I The following statement is a full description of this invention, including the best method of performing it known to us: 1 535-DLI I/110l OF T-CELL MD-'D CoITONS L TEC1121ICAL FIELD invention 'relates t-o methods. for the soeci-Fic depletion of activated T-1-Lvmphocvtes particularly zhose belonging to Ct-q CD4- subclass. Such activated Tlvtmanocytes e.g. CD4- T-lvwiohocytes, are imolicate. in a :9e number o. condi: ons in humanrs includinig multiple 16- sclerosis ari-d tran~sbTant reJCction,. Tn oatc u ar, chis '00:0 invention provides a rtreatCment in whnich. actiivated T lym;phocyvtes e.g. T-cells involved in a particular1 disease or condition are denleted while the non-activated T-lympho~yte e.g. CD4- T-cells repertoire is unaffected.

BACKGROUND OF I14VE-NTION The CD4' T-lymphocyte (herein referred to as the CD4' Tceill) ;s the. central1 player in the immune -system c'- *of.the Ielp" it nrovides to-other leukocytez in figtingq 20 off infection and potential cancerous cells. CD4' T-cells play essential roles in both humeral and'cell-mediated immunity and additionally they act during parasite *infection to promote -the dif-ferentiation of eosinophils and mast cells. if the CD4- T-cell population is depleted 25 (as is the case in AIDS patients) the host is rendered susceptible to a number of pathogens and tumours that do not ordinarily pose a threat to the host..

-eC_ us the aberrant: :uncrio of CDIY T-ce1i~s eads o autoimmunitv and o~her disease statLes (Swariborg, R.H., 9S4,, Cush, J.,and asiC 1988). Aucoimmune diseases iini whichn CD4* cellIs nave -been i-molicaced i nclude mu-Itln> sclerosis, rhe unatzoianr -xi: i s and auto cimmune uve't -3 s S-e gene ral1ly, an aberrat- immune resp onse in whic the immune system is I subverted f'rom its normal role ofF attacking inv-adina c p* athogens anda Instead attacks th e h-ost body tiissue~.

l eading to llesand even death; The Largeted 'nos 1 tissues vary between autoimmune diseases,~ tor exambie, in rultiole sclerosis the immune s vs.tL41 attacks :he; qhite matter of the brain 'and soinal cord. in rheurnatO~l arthritis the irrs-riune system Itak Lh yoiin I n ofAh oit. ct ivat ecI, ,'D4 T-ce'Is haveti also been A) implicaced in other ilnessas, includirg ree ctc Lio transazlant cissusad as arid in t 1 e ae .z-lopment *.CD4- T-ceilWl DLphomas.

ivsia'~ nto conditions causd by aberran CD'= T 2i cell activitzy are f ocusec. on seveal animal,.models' anda in particular on a number oE expihrimentallv induced autoimmune diseases. Research on these experimentally) induced diseases in animals is premised on the idea that I *.t goa zeen sn. W -C I areF re SOnsz2 oz severalI Ily ermn~ l vziucec a~~mi iess~ anials, including exzerimental .au~tojmmune e n c e ph a cmve±11 (EEcO'cr-a n nucd ahiis (Chand exieri-metJ a u rammule uveii

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04E !s induced by om_3' ani~mals acrainsr t!iwliin basic -r-otce (NGP, F comnonentr 0 ar. Wvnice ~crof U-he brain and tne sbinal cord) anQ ponuces zme same clini-cal s-vmotomS observed in .mu Ito 1 e sclerosis: *S demvelination and paralvsis. Proof c= -he value or mn'he A mdl sa ooa e model for multisole sc os if; 'has been provided by evidence showing that these.

conditions share a 'causative nexus: Sm-eInman and coworker s' showed that the. predodminant cell tyo e found in thebrain lesions of multiple sclerosis patiants is CD4 K -T-clls (OksenTberaJ. R -2m al., 1.990) arid that the Tcell receoro (the-molecule responsible fo r antigen recog2.iofl) as~oqcitedQ with the cells in chesE_ brain lesioins had he samie 3 -,nzino acid -b ind ing motif for an aeni recogni'ticn-as on th e CD4'T-cells re5SDOnsible 7or caus nexDer",mental aumoj-rnmune enceohalomVelitis (EAE)-j (Oksenberg, Ji.R. etial., 19931... All the evidence thu's suggests that the EAE model will be useful'in testing; -therapies for multiole Sclerosis.

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I VI Id o'- ~0 cveomen a::ese ciseeases as 're as nh~ik ranszanta ;on rejecd-_on (Swanbhora7 ±,933 Cob-o'. S at:ia -I 19 O3 SeJm_= n 3 CDr- c a b- d a I- to ti s~ C .oi~ Cse gen 11y S a n-m a- L I LD. 4 I -E t Z a :oa t ha t eaetinc apoes that at det n -te De TLD4ho tEI. -rsu a poock t-on micti. ob hc 1 sn rlso t ez he-C andcth s invlve inte ="cU an b'.'the. immun imnuaooprmie and hence isno benly suseptibl o uc ifecita pocrs thab teautaoroachbes tha deoestoy thoe CD1 T-celis ~tha ~oa o a ih e fctivel inovd nte-uo Th i s rethc-; -ff rea-cnent 1-:s n-oc yet been acn--evec. -1 s re-fo 07 c Lhc P~sn c- n:oro-ca ~e~nO c sC~Ca 'v cne po-puia-2o c: 0*S acc-azed CD, z- e' I -i saan IAou n:e:n a so t7u, -::cent CD-- C-L n SITRhr[AIRY OF THiE I1N~O Tn one asa-eC- tz4 oresent invenulcL oro71cZC ae tetnou bcv; nclautoanzage-il sbecliflc T-caiis, eszeciaiilv Eor examD±C acc CTD4 T- c elIS, can be S'De Ci C ally e1i min*:a Led vo, wh-!Ie leaving the ou iescent opulatlon a -cells especialfly Eor exampole unactivated T-cells C Thi--.s invention- thereore provides a ,q t-r e at-m e n u s e T cellimediated especially for exatnle activated CD4' -cell meda-ed aultoimmUne diseases 210 sucn as mu-il scle osis, dmtO. arthritis, saprcoildosis a.a au-o mmn uV~e~t vrusc di dsease arid/or inflammatory nooal disse Thii v-n r Lon 1asG orovice5s a aethoc. fo iniaifgote nd d u- res onsas caunsed by activated T-cells esrc2.ac1i ;-or example adtr ivated CDL Tcells such as rejec~ -o7 of transplanted tissue and organs in transolant recipients.- Furthermore, the present -inventionl provides a method of snecifically eliminating activated CD4' T-cell 7-C Vy accr .~uorec~iC -CJ.ls at. tcnC s-1t-e of aut i-m-mmune lesions otn" armful T-cel ivaoa. aserv =-rl.c5 -f lindit= Lna e -at~Os 0- Z: -rat n urovidce(Q: ID- mel oresent- -41et-ion.may" be and cariij~:.~ earlier treacmer-t or n 0 cLr Crlt2-3 ra S1C etn be OS The aese inen~zcn is also basedc on he disoez; ta a. artic--Cr protein: =nien, rered O)X--C Che=J arred to as z:-e O'K -W antigen) is soec-_fica-,I exu-r-ssedc on, z'e Gc 1 surizace or antigren activated

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cellsz especiall-v for examole activated CD4 T-cells

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Particular, usn the E disease model in rats,. this 0 antigeni was snown to be e prassed on the surface c f acti-vacer-. autoanti.. el-soec-i 'LIic CD4- T-ce-j,--ls orese.rit at the site of nlmmto (th--e son'al cord i nthi s di sease model) bur abs e nt on CD' T-cells .at non--;Infa nat sites. Frru LIhies exoression oi t:l7 an I en or-. te T-calls w, as 'found to occur 0- the davy orior co taoro cl~icalsins o auto-innmuni-v; toe-- excaression of this adie ecreased as ~h dseseoocressed. Th sejt-lyorepresSion or %I-he OX -4t antiaen and.Lhe transient nature of thi-s exoressiofl, shown for the first time in the present I 44 n -I 0 III :j VI F; to 04 'u a III c i 0 (41 0 III JI II 1 III a (V 7j '1 I C U 5 V) 'CS 'r 424)~ 0. a) C .42~ P *0 o-i 1 .U t' U 4 0 -4 u r.1 -I i It) IJ 42. U) U) 4- V ti 'ii U (V11) 0j q) 0 to f, 11 Cij U) -I I ltU 'i .1 .l a) n 0 1) r4) DI UI (1 (21 a) .4 4 k1 U) u4 1V: I/i (D -t u a4 U .43 i U 2U 4 i I '-4 tit I 1) 42'. .4 li 11 ,U I> 4.4 U )4'(1III C) .11 (v u II] 4 Il( 5 d( 0l 1-.1 C L 42O -I 2 r C)1 '1 C1U') '4I 44 1. V 'I'~11 (11 ''CJ U 3 l4 v: (31 4 l 1 I. 11 1-1 r*H In 110U .1 4) r- 4 'U I M42s'I ti) (r C 4 2) 4 U 3 Li a) r- U l In-' 43 .1 1 '-4i 9 1 01' C' tUr 1) U) Id r.4 4>C) II)~ 1 2 1 4 0'1 0 j3 I4'-4 U 1) 42 ~T o) '44 U di u4 U).2U 2 U C 1 Cl 0- 4-j IJ 1) 6, (V .4 14d .4 i J (U II Ut UI 1i 44 2 C C 0 1) It 4 O C) 4 5 4 'I i C fj 4.1 0. U- fl LI C U C 4 I *C (It I'l 1i )14) 11 C) U)r) U -I C '-I 14) Ii U 'll .42 T U (V 044II Ci l Uf f-1 j M4 0:1 p 0 *0 0 C*,I (-I O aii~~ O-0 0a e cae Sea ID No.c ac g an~~l. Ic ~ermninanzs of ox%-4 o 7iart are accassibl cosec c he Ln OX-4 i ce se on all S -l n SiLc: finic; a S= taa7 aritpr On, a -tctiar cellI type doe n opr.r flasis. c Oa teran utic app Ian wiicn 1 0 t- hr a oac* :--IaI c= u. imany an-* c~nc aY' c-a su=ace~~ n s as L 'ae s o-flo t-rapy- c sa -as -~an ibodv ra-iseq aa t ns che OX-4 nrocin.Qa c-1OL~ct-C. oaCtL a nii~te VZ oo er t b. -Icen ac~lva..-d CD4I- T-celi's.

Thiis discoverly _.Mlies that the OX-40 antigen 1sr~fl intrnaize byCD4T-c ells. Additional researcbae Inenl~e b D hae i.

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0-~-se ann.n;&c rOc. ra-inn a 1 I On of a n jc n ac iVa:e a T-cells can !De Od-ee zr vlvo by., conjugaz~ing a= szecCfc biding agent such as an an'tibody raisedt against the OX-a40 ant 4gei -with a cytotoxin t~o o-oduce an munotoxin, and -adminisuerincr hnis jnmunocoxin to a hoCs-. Tn this anr tne anLIbOdyV binds to tihe OX-40 antigen on the sufc o-f the -activane-d T-cell-. ITncrnalizatian of the inmurkotoxir.

ftresulits in the c-vctc~tO beinc_ taken into cell, wic o;roduces cell1 deat-h. {e-nce, adinistration of this *irmunotoxin to a host suffering f ro0m activated T-cell (eg CD4- T-cell1) mediated i nflammation depletes (or otherwise 5 inacti vates) the activaced T-cell -s especially for examPle activated CD4- T-cells at the site of inflammation or other sites, leading to amelioration of subseoruent.

inflamnmation and/or otherz clinical si-gns of disease- A further aspect of the present invention is rherefore a meth od of treating a pat ient suffering from a condition mediated by activated 'P-cells e.g- CD4- T-cells, whic h comorises administering to the patient an effejctive amount of a' specific binding agent which can specifically bind to a polvroeut2-de as described above, eg an antibody conjugated with a cytotoxic agent wherein -the antibody recognises and binds to the OX-40 antigen present on the surface of the T-cells, especially for example activated CD4 T-cellszretno to: dscn :CZ' ~ac __on -ce s e- 77 -Da 7-Cefl ex<r=ess an ox-ao aritiae2 inla Inu-man Hoszi and a miechod Of irn _.bi ui n relapsinlg autoimmule _Jn_'am.nazuion Ln a Datient suffering froma multio-le scle'osis, which methods comprise admtinisteriLE an effective amount of a soecif,ic binding P .7 in particular Lh4 specific bindliflg agent used inr these me th1 1ods is an antiboy co -uaed with a cytonoxic agent (aniwmnoo~il) heein th anibody recognizes and binds to the OX-40 antigen present an the surface of the T-cells especially examole activated CD4' T-cells.

Alternatively the method may employ aspecific binding agent which comprises a Fab, L(ab')2, or Fv fragment of a -monoclonal antibodyv -capable of recounn-sing Ox-40 antigen when expressed on the surface of T-cells, other specific binding agents useful in this method are immuno globul-ins capable of cytotoxi ffect*ori cells bearing Ox-40 onl 20 their surface or any specific binding agents which can fi X, COMDlement or mediate antibody-deflndalt cellular cytbtoxicity such as..a specific binding agent which has or is linked to structure, characteristic, 6-f the Fc region of an immunoglobulin of murinfe typje TgG2a or human type 25 TIgG1 or I gG3.

The methods of the invention are applicable to any condition mediated by activated T-cells especially for eaieacr-L%-a teCD 'T C S i- c ul-J 7 -ulcIn s c l e c2i, s a r' C m ult: acs, s r- SCmacoCCS a r a-ucsoc mmune uvetis 1v~ob~ma an rjecrion of a t:ransinlanted or an or tissue. Addit ion al7 Condto~ 4o whc ti 5 me-hod is applicable include cra-ft-versus-host disease or reaccion and nl~mtr bowel disease.

-cells can be acti1vated b y fo_- examlr~e antigens,sunerancicfl-n, mitogens, or monocionaL antibodies.

The methods of treatment set forth in he urecedJing p aragrab'n will Preferably be perforrned using specific binding agents s c h as monoclonal antibodies, or _ragment.S t-hereof, which can be raised using the polypeptides o f thle invention. in a more preferred embodiment, the mionocio~nall antibodv will 1be a humanized monoclonal antibody. In alternative embodiments, the method 1il utilize a' cytoto~xic conjugate,, eg comprising a Lragrment such as a'Fab, or Fv fragment of a M 2 monoclonal ant-ibody conjugated with a cycotoxic agent wherein the fragme nt of the monoclonal antibody recognizes the OX-40 antigen.

Tb 'Vs ivention also encomoasses specific binding agents such as monoclonal antibodies having a speciEicity of *binding in cells to substantially only antigen activated T-cells especially for example activated CD4' T-cells. In.

a preferred embodiment, Lhe specific binding agents can

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Parzicular suecifl.c binning agencs soec-4fically bind to human Ox-40 of amino acid secuence encoded by the coainc region of nucleic acia sequence Sea 1D No 1, when said 5 ox-,i0 is present on the surface of activated cells.

These spcfcbinding agents suitably comprise an r antibody binding domain and are preferably monoclonal antibodies or binding frarments tnereoff .As. mentiojued to above, the antibodies will pre ferably be at least partially humanised, and so most referably comprise a humuanised monoclonal antibody.

"Specific binding" refers -for example to specific noncovalent mo-lecular binding such as that between an anitibody-and a corresponding antigen or hapten (its "binding partner") and also between other specialised 'ri-ndinar molecules and their binding- partners-.

"At least partly humanised", relating to antibodies and their binding domains, means that for example embodiments are contemplated in which only the constant region (CH and CL) may correspond to human polypeptide: alternatively, both the constant and variable regions may be "humanised".

Another aspect of this invention provides a specific binding agent such as a monoclonal antibody as described above t.niich furzher coniorises a moLe'ur i-d' ec: cova len t v conj ugazted, cytortoxin. Anzbodv-ctotoxin conjugates (also known as immunouoxi1ns) aZre suizable o use in the me-nods of-treatment described above.

Examples of such specific binding agents include Fab, F Cab'), or Fv fragments of a monoc lonal antibodyconjucat-ed with a cytotoxic agenL.

too.. F or. theraoeutic use, th specific bindinq ageni-s ofE the invention are suitably administered in the form of a pharmaceutical composition which include a pharmaceutically acceptable carrier. 'The carriermay be solid or preferably liquid carriers such as water or saline, which are conventional in the art.

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S A further aspect of the-invention comprises a method of *det,~cting a condition mediated by-activated T-cells, e.g *too 'CD4- T-cells,- in a pat-ient comrprising;.contacti;ng a specific b indi ng agent as described. above -with. said T-_ cells and quantifying the- level of 'activated T-cells.

~Suitably them mthod is carried outoabipyspl from theatnt such'as a skin 'or intestinal biopsy samp Ile or a blood sample from a patient suspected of havingT a. graf t -versus.-host disease' or an intestinal biopsy sample from a patient suspected of having an inflammatory bowel disorder, or a sample of cerebrospinal fluid.

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sc cu~e f pn riserr v :s a me tnnaoe; Qetetin a~ ~la~atrv cnc~inmei-azec by a tivated T-ceils esrecially for examnle activate--d CDA- T-cells in a uatient_ bv obt-ai'nin'g a suitable bioosv sam-ple ftrom the patient and then aruantifying the percentage of activated T-cellis especil± 1 ly fcrxamoln activated CD4 T-cells in the bi-zcTsv samole -usi ng a suec.fi bi ndi ng agent such as an antibodv that spci ia1vbin'ds to the OX-'0 antigen.

Ot~aspDects of the present invention include test kin 0 for detecting conditions mediated by activated T-cells especially for example activated T-cells, and treatment kits' comprising antibody in pharmaceutically administrable forms and amuounts with suitable excipients and containers.

.e~ S C CC C In particular the invention provides a kit for detecting *see.:.

4 e a condition mediated by activated T-cells e.g. CD4* TcellIs in a pat ient compr ising a specific b inding agent as described above which is-labelled.. Preferably the invent ion provides a 'kit for carrying out a s pecific binding assay for detection or quan~itation of an analyte that comprises a' polypeptide or a s-pecific binding'ager-t as described above., wherein said kit comprises a first: reagent comprising a specifiic binding agent that can recognise the analyte, a second reagent comprising a substance that can bind specific ally either to the analyte or to the first reagent-, and a label for the second reagent.

nsu--a Eis remaen -cp s na s -c e c analvte, and Le second re age comorlses a iaeled anticlobuIiis_4cro h is ASternat'v ly the first reagent can c omp rise a n immobilis'ed suecir-c binai-ng reacenc taor -he analvte, then the second reagenE is. a speciltic biinding.agenc tnaz car. bind to thli analyze the analvte is also bou-ad o th'e t isr-reagenL.

In a further embodiment wherein the second reagent comnCrises a substance able to compete with analvte for binding to the -first reagent.

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SC C S 1 Foruse rnhismetodthespecific binding agent may f urther compri. es, a label such as a radioactive label or -a fluorescent label and these specific binding agents o~m~ frthr apec nfthe dinvention.

-he specific binding agents of the invention may be clse ad~eqenedinthe usual~way. Thus the.

invefttion ftirther provide s a nucleic-acid sequence elcoding the amino acid s e4ueneo sp~ecii idn agent. as described above.

Reombinant technology may be used to prepare bt h pol: ietides and the specific binding agents of the

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*Sa *0 -1 1 3 invention. 7hs ap rcnriat ex:.:zrssin vecrrs, Lr~S~C~tC hstorganl.5ms an mezlnodz of prepaaz-on whh incl -ude culturing a -ho s z:oacanims'-form furhr aspects cf- the innvention.

The 0X40 binlQii--g acent, for Instance an anti-0X40 monoclonal antibody, can be admininstere6 to those j atie zs suf fer Ing from a disease medaiated by activated T cells e.q. ara Ft--versus -host disease. TnamountL admiiserd will depend on the amount recuire6 o produce an imorovement, eithe 'atal or oai h DatL'Cft's symptoms. This wLl-l de-oeno not only o n the severi41ty of the condition 'and route of administration but also on the,'administration. of other theraopeutic agents (eg glucocorticoids,' cyclosporine A: prednisolone). The 0X40 binding agent may be injected either sytemically (eg intravenous) or locally (eq intramuscular).- As discussed else~ihere th6 0X4.0 binding agent may bp coupled to a toxic substance for maximum ther apeUtiC. _fCt.

and.Graft versus Host.Disease

(GVHD)

In the c as e o f GVED immunocompetenff T cells deri ved -from the donor tissue or cells attack reci~ient tissue Iincluding skin; gut and !liver,- which, 'are severely cbmpromised in t he il ability to carry out- Lheir riorma%.

functio. Suchattacks, if not controlled,caledt *death of the patient- The therapeutic agents of the present in vent .ion could be used to block the activation *OX -40 and GvHD: diagnostics The cresenc invention can be, used d-Jcagnostic tests and prccF~au es n vitro.- For instance r_'e 0X40 binding agenc can be useco eetieLe of OX4O- T cells -i *abioosv samnle from a oatientc. Tne biopsy samole may be a tissue samnple or a sarnoie co: bod. Mononuclea.. ce-i-Lc a-e -solated from Lhe blood or tE:ssae accord ing E~ standa rd techniques (see Practical I,,Mmunology. L. Htudson and F.C.-Hay, eds- Blackwell Scienzt;flc Lpublications, *Oxford) and stai:ned with aa anti-OX'aO antibody or 0X40 *bindinq agean t fu11s ioan 'Drote-in The nresence o f the 0xal0 1. .bnding -agent> is then detected with an antLi-globulin *reagent ~coupled with a fluordchrome such as fluoroscein isothiocvanate or phyqoerythrin and the number Of oositive cells analyvsed o n a flow cytorneter or by fluorescence macroscopy (see E~g Pr acticalPFlowCtl ltY Shapiro, HM_, ed. Alan R. Liss, _New York; Practical- _m~ooy. Fludson and F.C Hay,, ads. Slacwel Scientific Publications, Oxfqrd) -Al-ternatively the tissue sam le -is processed ror immunohistochemical stainiing by standard techniques (see eg.

*Immunocytochemistry: Practical Aplications in Pathology and Biology. J. Polak and S. van Noorden, eds. Joh~n Wright and Sons, Bristol) -The 0X40i population of cells could be further characterised by two- or three-colour 005 *ego York; iuovohz~LV Pa~oovand Eioo ay o J. aoaK an s. an doren, eds.

John Nricht and Sons, Bristol) Detecction of OXAO cells 'can am n th E ediagnosis and manareen of diseasescaused by a-i-,at-ci T7 cells, in F-aimmar.ory nowelI disease and G-vD anu itay bDe use d Cor±o ne course or the di, ease: in increase In crie! conrtion of OX'40±. cells O would suacresz a wors-a- ng ozL the disease arid may ncae n ~Q e aed to increase the dose o-Ft e a D eU ZI C agent being ad~ministered, wn. Ie a decrease'L 'he nooortion. of 0X410 cells would suggest an irno-rovement an h ru s indicate a dmin u t o n I n -re a m o u nt o f th ra pe UL Ca 6e nt being J, administered.

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5* 5505 5 1 Determination or- the levels oE0'O~e 1 inte lo of Dpatients at rlsk of GvHD (eg followin alogenic bonemarrow transplantation) may allow on, to predict the i mminenL on-set of GvHD. Early adrninistrati 6 n. of immunosunressi-ve agent'Ls to -(ontrol GvHD-will improve the likel-ihoodof successful treatment.

The-present nve n t ion can be used iJn diagnostic test n procedures in vivo. For instance, the administration of, an OX 0 binding agent coup] 7 ed to a rAdiois'otooe.cal be used for the pul POs of immunoscinti-graphy.' bccme more re="i V -0,~Zr OF THlE DRJNWINGS snows dot pcs from a z Iuorescence acciv~aued ce-1so r C2nq (FACS) apoarartus showinc tissue..sn-acific dual exnression of RT7.2 and n- MRC GX -40 ge ni' :,.mmoc ,es were i -olatoCd Ltrcm -4ie various :-ssu- *cocarmencs des-tnarcea n r :c Cre sun an cst o of E:AE. 'The cell were stainedi with Lthe 0--0 antibody co njugaced rnoL fluor'esce-in isothi*oc-,va:1ate- (7TTC) displayed on the x-ax-s ana couiterstain with a R-uh'ycoe::-.thrif nC-- conjugated to the PRT7.2 a n tibodydisplayed on the y-axis.. An isotype matched control antibody was used to draw the oruadrants for both the FITC arnd 2E conjugated Abs. The OX-qO antibody was *positive con the. donor- T-cells isolated. f raimthe spinal cord,. and 8, 2, and 1.8% posit-ive' for the donor cells.

20'. isolated fErom the CSF, spleen, and blood, respectively.

F ig_ 2 shows dot plots from a -FACS apoaratus show-ng a time course of OX-40.exnression. on donor r-cells isolated F rom .t he sp-inal cords o f r a ts wi th ElkE.

Lwhc~swr solated from the spiinal- cords of rats during the time course of disease- The cells were stained with the OX-40 antibody (FITC) and counterstained with a PE conjugated RT7.2.antibody. An isotype ho oi C_ eC course ffc ec ao olt is _J-JCa:edc n trie tou of granh ch Ln Qsease score 0 clinical signs and diseasescore is pro-vided in the2 metn~oas secr-ion under 'Adoptive T of RAEo is s.ow inornns next to the cav of disease. m e erenag ofRT.2cells we= 11, 73, and 52:' resDetlV! 'For Days 93 n fe'' e :ccivel 1, 2, 3, arCLaue re- Po onset. Tne OX-40 anztibody was 3~uos iva for the donor T-ce1is on, Day 0 (day before onset) and IS, and positive on davs 1, 2,3, and 5 respectively. On Day EARE score of +i me ans the animf,-al had Enmi ma Clinical signsz o- aaraivs's (less than 1) hut was not ~-Th completely wellI Ficure 3 shows TCRV13 express ion in OX-40 positive and negative sorted populations isolated from, the spiriacords o f Le~wis rats w it h 7 RA-E. Lewis rats. were'actively imunse ith mvelin basic proteinin CA Spnal cord lymphocytes were isolated at the -onset of disease. The cells were immediatel lye fe h X4 ea-ion, the RNA was isolated, and. analysed for W8 T cell 'receptor gene usage., !n the- bottom two panels the cells-were sorted with the FACStar (Becton-Dickenson, San jose, GA) while the top panel comipares the unsorted popula tion.

The unsorted cell population was stained with the VPS.2- FIETC antibody and showed a similar positive percentage-as ia _z 'cws anQe ccsconse C= nslae fro :Ih sninal co-rd and noe r-azd wereacrrelv rntun sec withn MEP C'F and n rn Cord, lvmnocvnes-and imn none c-;1 wer ioa"red aa Th cels.wer nroOX-4C' and o Ofrac. io s, cuitured Zor ayvS, and were :s u~la-4 th~e cnrresont7-,am: cren -he u-sorrced *sI-iial coard ooo -L-ar on ,as culI-ule inT for 5 dayvs andl asVen ncII The cei is wer -cu- arenwLed es Okdar' -O~LC~RC) anid am-iren 3~E] TI rmnI die was adde later. Tii.picac:e wells harvastEOQ oro gla~ss f.e i b rfi ers r aif+_-e r the label was added. All or thie cell types (except: the unsortedn obulation) were s ci m uI t ed with Con A as a control and showed auoroximacel-vr .100, 000 CPMs. for each- group (data no t shown, Fig. 4 Cr5S a grapn shwlg doed ndedt inhlibi tion or anticen sneci~c CD4 T--ce-ll prbo1 ze ra t 2on -b OX-'i0-da&A conjugate.. Varying concenran 4 or's of, cae immunoutoxir- was added to a conszua:nt arnounu. or Fl T-celis, ar-Iuaen- oresentling cells and ('yellin basic Orotein anrigen). The open bars (to the right of the graph) show the proliferation (represented by -thym idine incorporation) of these T-cells with and without antigen with. no immruotoxin added (Pos and 1Neg *4d a 1'

S.'

F d Fi G3 are toarapns showln-Iia nose i.z o- ~n o z a n I en s n oac fc C D -c ei zole a o nn or the nX4-xocc n -aate mconcnMons o f X-']-xotxa was accea Lo a cc-ouscno AC, dn MB.Teocen ba-rs fto <alt of che 7_raphs) Z'w tacN incort-ora ofF these T-Ceils a nd wqItCh o u- ant;ican (Po ad Nc etlv The assay was carrlen out a (Psan.Na-.

15 Fig, 7 Is a graph S how i n- do Ise dleoendent inhibitionl o'r PPD antigen spacia_-- CD'4 cell pol -f er-at ion by the OX -140 -dg.A conjug='te'., Varyig -cnenrations of the OK-140 o imm-notoxhf was added to a- constant. amoo of 71 T-cei.s, =trtiaen presnting tells-(?L and PPD (an~ti.eFn-. heoojen iars (to- the _In th aa)shw h orol irreat-ion_ (raursernt _d by H 1 1t sd ,rlcrooatjn) o Cels wth and wi--thotanle (-Dos a-nd Neg Te~cie The assav- was carre ou a 200 ~ivohrrm 8Baetw2o5-lt Fig. SA a nd i.82retodtnosfrom a FACS apparatus showing characterization Of: lympihocytes isolated from the spinal cord of rats that had been d* t ra d wi th a 400C pq dose of CX 0-dcA GAP) or unnlraatec racs U(Fic. 63) F1 'MaP Szeci__l CD"- T-cells were trans-Ferr ea into0 irradiated Lewis recijoients and was given'on the -same day of transfer. Thte transfer Dooulation was detected by the RT7.2 antibody conjtigated to PE (represented on the v-axis) and cou-nterstained with tne control antibody ants -rat

IM-FTTC-

1t) Figure 9 shows OX-a-0 imu~~3rreatment ad-inis-ered on the fEirst day OL disease onsezt. _71 MBP snecific T cells were transferred into 8 irradiated Lewis rats. On the f irst: day of disease onset 4, o-f the animals were "a treated with 400 ,ug of OX1401 immunotoxin and were compared to the untreated "Conttrol" animals. After t* reatment, both. gjroups were scored daily until the clinical signs of EAE subsided. Each point on the graph represents tile, mean- clinical score of 4 -ariimals f .or th e created an~d control groups. The clinical score~.were treated on the same scale as described in previous figures.

9 a' 604 a 'a' Figure 10 shows effect on donor positive (MBP specific) T cells isolated from the spinal cord of- animals treated with OX-40 immunotoxin. Irradiated Lewis rats were injected with F1 (Lewis x Buffalo) MBP specific T cells x 10' cells/animal) .In panels A, C, and .E the animals were used as "controls' and received encephalitogelic cells alone. In anels 3, D, and F the animals received encephalicogenic cells plus a single dose of immunotoxin i.p. (400 pg/animal). Panel B shows the effect of the treatment given on the same day of cell transfer, while panels D and F show the effect of treatment given the day before disease onset (4 days after cell transfer) Al the animals were sacrificed on the first day of disease onset which was 24 hr after treatment in panels D a-id F. The spinal cord lymphocytes S were then stained with RT7.2-PE and the percent positive cells are shown in the top right corner. The total number of RT7.2- spinal cord lymphocytes are also shown and was calculated by multiplying the percentage of RT7.2' cells by the total number of lymphocytes isolated from the spinal cord prep. The EAE clinical score is represented in the top left corner and were the animals were rated according to the following scale: 0, no signs; 1, limp tail; 2, hind leg weakness, ataxia; 3, hind cuarter paralysis; 4, front and-hind limb paralysis, moribund condition. In this scale a haif step means the animals were in between the severity of the numbered scale.

Fig. 11 shows the nucleotide seauence of the coding region of the human OX-40 cDNA and the theoretical amino acid sequence of the human OX-40 antigen. These sequences are encompassed within SEQ I.D. No. 1 set forth in the accompanying sequence listing.

DETAILED DESCRIPTION OF VT17 INVENTION The oreserit invention orovides, for thne firsz time, a method of eliminauing undesired immune responses caused by antigen activated T-tells especially -for example D activated CD4' T-cells. Without wishing to be bound by theory, the inventors made the invention after making f indings- suggestive that ,a cell surr-:ace antigen, 11e re T1 do: termed' the Ox-4O antigen, is upregulated solely or r preferentially on activated.T-cells, especially f or sod#0 example activated CD4' T-cells found at the site o f $@ee *ifaation, and that this cell surface antigen appears.

to be internalized rapidly. Based on this discovery, a theraoeutic method has been develooed which utilizes antibodies which' bind to, the OX-40 protein (herein termed off% OX-40 antibodies). li nked t o cytotoxins, to destroy cells expressing-lthe OX-40 antigen.. This type of therapy will be extremely useful-because i t is targeted only to 0: Aa activated T-cells especially -forexample activated CD4' Tcells-and leaves the rest of- the T-cell repertoireintact.

Activated T-cells especially for example activated CD4 Tcells have been implicated in a number ofE antigen activated- autoimmlune diseases, including multiple sclerosis, sarcoidosis, rheumatoid arthritis and autoimmune uveitis, as well as in'transplantation.

rejections- (Swanborg, 1684; Cush, and Lipsky,.P.E.,- 1988; Caspi et al, 1988; Cobbold, S.P. et 1934.1 CD4" T-1ceil lvmpha) ,mas nav.E also been sno-wn to have an acr~va _e phenotype (Gootenberg, J. E. ez: 1981). The oresent invention orovides both mrethods of diagnosis and methods of treatment for these and ocher conditions mediated by activated'T-cells especially f.or example activated CD4- T-cells. More particularly, and followina the descriotion of relevant materials and methods used in this invention, experimental data B*see, obtained during the development of the present inven:~o 1#3. is presented. These data demonstrate that the proLtein is exclusively expressed at the site of autoiimmune -inf lammation' in rats with EA.E on the surface, of myelin basic protein (MBP) activated CD4' T-cells. I t is further shown that the proliferation of MBP activated CD4* T-cells can be inhibited in vitro using an *ant ibody conjugated. with a Ricin dgA cytotoxin. This 4 inhibitory activity is shown not to be limited to MBP S Bacti"vated ce-lls,, but a lso to-be effectiveli'n inhibiting the proliferation of CD4 T-cells activated by other.

antigens, including._an ant-igen -de-rive'd fo yoatr tuberculosis.' The -OX-40 antibody,-cytotoxin conjugate is shown to be effective in vrivo,- use of the conjugate is -shown to inhibit the clinical development, of EAE.

Fol1lowing-this, -the cloning ofL the hu'man OX-40 homolog is pDresented along withi the production of monoclonal antibodies to the human OX-40 protein.

Various examples are presented showing the application of, thne 0resnt inVenti10. Soeci _ca lv, 7xo~ 0oreferred qiethods producing thc human CX-40 cDNiA enabled by the oresent invention. ExamnL 1 .es e methods of producing puri fied human OX-40 Dro .ein, and Example 3 describes the production oE monoclonal and polyclonal antibodies that recognize the human protein.

Example 4 describes the oroduction o: immurotox~ins, based on these monoclonal antibodies, thar are suitable for used theraneutic use in humanis, and ot:her antibody conjugates suitable, fo diaanostic use. Example 5 describes the use of human. OX-40 monoclonal antibodies' in diagnosing activated CD4 T-cell mediated conditions and Example 6 describes the use of the immurnotoxins in therapeutic anplications. Example 7 describes kits for the diagnos iS and treatmuent of activated CD4 T-cell mediated.

a I conditions- Chas: MATERI!ALS AND METHODS Animals Lewis and Buffalolrats were obtained from Harlan Saue-Dwey Inc., Indianapolis, IN. Telve week old Lewis females were ~bred with 12: week old Buffalo male rats ~to generate the FL Lewis x Buffalo hybrid animals.

These F1 progeny were used at-8 to 12 weeks of age for MPP immunization. The rats were housed under germ-free conditions at the VA Medical Center Animal Care Facility, Portl1and, OR a ccording to institutional guiidelines.

Selection of MBP Secific F1 and Lewis CD4' Lymphocte w~n .tvei basic Oroteff 7 MP Detail--s of tns rocecur wer','e described earlier ('Vardenbark; ezt al., BriJefly, a lym.ph noe 1e suspension was i.ncubated with MEP (30 pg/nil) in RPMI 1640 with 1*i autologous rat serum. After 3 d~vs at 370C in a 7? CC), atmosphere the cells were cult~ured in 'RPmi w'ith horse serum and The T-cell lines were maintained in this medium uftil ;The rate of d'v-sion slowed. At this 042n (-4dys fe M2? stimulation) th'e cells wer rest imulated witi. 10 pg/nil of MBP presen ted by irradiated' Lewis thyvmocytes, and-,subsequently expanded further in IL-2 containing medium-

S

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5~ *5 .0014 Adoijtilfe Transfer of EAE Activation of the F1 or-Lewis T-cell lines-for passive transfer of EAE was carried out in 10 cm' culture. dishes using 5 x 10' T-cells, 1-00 x irradiated, A7C, ano.- '10 pg,/ml MBP in 10 ml of. medium.

M After h"ree diays of activat-Lon' the blasts were -counted and 5-10. x-10' T-cell blasts were injected with the associated- APC population i.p. into-irradiated (600 rads *for the- F1' into Lewis t ransfers) or non-ir rad iat ed naive Lewis ralts.- The naive allogeneic recipients were irradiated the day before adoptive transfer. The recipient rats were inspected daily, and the clinical signs of disease were recorded and scored-as follows EAE: no signs; -1 =flaccid tail; 2 =ataxia, 3 =hindquarter paralysis.; 4 =acuadriplegic/moi:ibuld.

Cel CllecionCereros~~~ fl 'd (CSL) was collected by perfo--mjig cisterna magna ouncture using a 27 ga x 3/8' needle with 8" tubing (Abboct Hospitals, Inc., Chicago, IL) The CSF was diluted in RPM! 1:4 and viable cell nu-mbers were counted- On average, 100 p1/rat were collect-ed. Samples were excluded. if tuhe RRC/WEC ratio* 0 049 *exceeded 2:1. The bi6oc: was o Lalnned by heart punctu.Ce 0. and the lvrnphocytes were scaae nicoll-ii-ypacque as ***described by Kruisbeek,,A.M., 1992. The spleen cells were pushed through a wire mesh screen and the RBCs were lysed by the NR 4 Cl method (Kruisbeek, A.M. 1992) Spinal Cord Lvmohocvte.Isolation Spinal cord mononuclear cells were isolated following a modified version of a published protocol (3ourdette, DA.I. et-al., 1991).

Got.* Sb.iefly, spinal cords-were isolated by insif f lation, 4shed 3k in RPMT in'order. to remove, any contaminating -blood cells, homogenized, and then passed through a wire mnesh screei. The cells were then washed and resuspended in isotonic Percoll For each individual spinal cord a 10 ml step gradient was poured into a 15 ml conical tube. Each step gditha10%(2 ml), 809; (4, ml), and 401;(4 ml) isot-onic percoll and the cells. were lay ered as part of the',80% fraction. The interface between the 80140% Percoll steps was harvested and the cells :were directly spun down and washed. The.

r-esci I s zs _4ic occulacion. s ess=ct by forlaarc scatter. Avica', Vi eld ofrnoove obane rom the scnlcord oE anriais with was usually 0.5-1.5 _x 10' cells. Ly-mohocyte recovery was fairlv consistent throughout the disease time course, and decreased during the last dav of- the -recovery phase of: -=AE to 1/2 or 1/3 of the maxima! cell nurnber..

0 luesec acivated cell sor-ing (FACS) analysis Fojr the dual fluorescence analysis sho-an in Fig. 1 and 2, th e antibodies used were the RT7 2- PE Ab (Pharrningen, La, jolla, CA) and theiMRC OX-A0-FITC ;Ab (Pharmingen, La jolla., CA), All the anaivsis was oerformed on a FACScan .00:015 with the FACScan Research Software Version A (Becton Dickenson, San Jose,,CAY operated'accoryding to the manufacturer's.:instructionS.

Anticen pcif ic Proliferation Assays, -Proliferation assays were Performed in-96-well plat Ies as describ~ed previou'sly (Vandenbark, A. et al., 1985). Briefly, 2 x CD4' T-cells and- 1 x 10' irradiated thymocytes/well were incubated in RPM! and rat serum along with antigen an d varying -co ncen trations orf_-he 25 imnmunotoxin- or the toxin alone in a 200 p1 volume. The cultures were incubated for 72 hr, the last 18 hr in the presence' of 0. 5 Bq 3 HJ]- thymidine. T he cells were harvested on to glass fiber filters and [IH)-thymidine upzak~e was assessec vzuc c.n' aer Caiculaced !,ron trnja heS r .eic~te2 wells vaz ed <10, from Zthe_ mnea n y OX-40 IMiinotoxin inhibiteon or AE Naive Lewis rats wec e injected with an enceplalilogeflic dose of MB P so e C if-C C T C es -jcted in one flank and injecte.

wich the imunotoxfln aL 7-h same Lime in zhe opoosiLce fan. O Criginally a dose cucre for th immuntoxin was set un an-d the o'rtama- dose asfound Lo be between 300-500 P9/8 week old rat. As. conzerols the sama molar amount of the, toxin alone (dnA) was given to aniasi parallel.- .*EXPERIMENTAL

RESULTS

One of. the keys to, understanding the mechanism(s) by' which autoantiqen specific T-cells destroy self-tissue is Sto study the difflarences-associated with an autoreact-ve T- cell- at the site of infladmmat ion versus a 2 noinlm aoYsite. T o thi s end an exuermeritaI model, system was set up Jin EAE to dtec h cls ta as the disease in vi vo.

EAE can be induced by the adoptive transf-er of in vitroactivated MEP specific CD4- T-cell-s into naive recipient rats. Four days after the transfer the animals start showing the paralytic signs of E. To allow detection of the donor population within the host,,MBP specific Fl.

neW.2S x Bu:JO -ce11s aez.72 S t e w snd an o r taIO express Llei Zar 1t of anr e o olf the CD45 cell surface mol.cule These aleic varaants are termedi RT7.l (Lewis) an~d RT 7.2 (BuffEalo) The IRT7.2 allelic, marker cani t-ee eor- be used to detect the T-cIl1i Lewis host: 'r ecause these cells. express both forms oi th e allel e 1L7h r=i r .s-ats on-l- 1 exoress the RT7.1 form orf t he allle CD4S ml icule is exprassed onlv on' -luo tes anLc constitutes approximately in's of the total surfvace P~otecn ig. 1sows t'aLt on tne first day of disease onset 501a- ofteBPoe ic tasered population (PT7.21) was pas it ive for -the activation marker OX-40 at the site of inf lanmat an so'rfal, cord)7. but the transferred 'opulation was negative f or .this. cell surface antigen at the acin- inflAimatqry siq tes (blood arld spleen).- This suqges ted'Iat .the OX-40 antigen was e xpre sse d oi the cell surface .:of autoreaCt-ive _CUV T-cells -upon antigen *recogna.tioh-fli if vvo, -because the IMBP antigen -is -present on the T- I& s in. the spinal cord. but not in the, -blood or spleen. _The highest .exprqssion of thle OX-40 an~igel on the donofr noculato isolated ;from to-the spinal cord was on the dav ba fore. c! inica -s igns of EAE and as- the disease brogresd this cell surface molecule went away (i.2) The Vfl8.2 T cell receptor (TCR)_component is believed to -eii~ 7scs Wo 7" be p-redam~~ranC v'.gre pr__ aso enen .l-tcgenic epope ct -mve-1 1: baslc protein (3)i Ljewis 2.-ats. eC1 rat s ciey mrr sed with and OX-140 oositive and. neaative ce'lls wer&e analysed forant i cen reactlvlty an-rd 7CR .Vg utilIisation (Fiq 3 and 4) Sorted OX-40* T cells isolated from t_,e spinal corn- were nighiy en'riched for exoresslon o-f the V88.2 7 cell receptor component comar-ed toJ- OX-4 0-o notc spinal cord popula.tions (see Fi c 3) 7icure 4!L ows that wh-ereas both InmBP andi ODD reacctive T cells showed enricthed resorises in: the OX-10 -racto of -hdning *lvmoh, node, only N13P rea cri v a T cell s- were_ found in the O-Qflzaction in Urhie. inflamed CNS. -THese datademonstrate the, selective ability o~f'the .OX-40 antibody *to mark the autoantigen reactive patho'enic T cells :-within the af-fe ct ed t-arge t-.or Igan in EAE. These data s strongly suggest.,that A4 stpecif ic: T. cell's can be ilaed and harctersedatlthe iteof.inflmmaionwith the'- OX-O antibody, thereby.diagnos,-ng the cell t roe directiy involved with i nflammation caused by autoantigen recognition. The -data also in'olv Ch-:isolation o f cells will be useful-ini identifying V18 biases and autoantigen specif ic c e 1 s w it hiJ-n inf lamed tissues even when, the antigen specifici-ty is unknown.

Therefore, the OX-40 antigen is shown to be expressed ex *vivao on antige-n act ivated CD4- T-c ells And, furthermo re, CCIs a Iiaaor~S1es 'here :ne an-znis eseo:expressed o~c a: nc-1-infla-mna or sites in the azsence of ancien Ths euls{ J tra-nsient nature arid target organ exovession of the ox-ao mark-er) sugested tha t OX-40 may be a dicacnosti c marl-er a na a suitable cta rgetr fo r antibody zmedlates d1eletion ofactivated aut-o-ivmune CD411 T-cell-s.

Tedee'on of .salecc'iv-e subset:s of -y-mohocy-es can be mediated bv antibodies' or other- binding prot.elns I.r V.:vo. ThIs. can be done bahe v choo s ing an antibody or other binding molecula which upon binding to cells ex ~r e~ Z :s g OX-40 will then act:ivate other effector cells or urote-ns o-f the -immune system to destroy the targetted cells: examples of this include lysis of cells via act iIvation 'of4 the comblem-ent cascade or via' triggering of ntibodv-deoendent 'cellular cytotoxicity (A DCC) (see H~ale -t a! 1583, bilood 62. 873-62.:Gre-=n-wood et al 1993, Eur..

J. Immunology 23: o03114 by -podif ving the antibody or bindin-rg -m oledule such th~at a ,toxic aceat is which will kil I :he'c~ll uoon binding. and inaestlon.

it is possible to modifyv binding proteins, monoclonal antibodies or fragments thearof by a varietv of means.

For instance, it is possible by means or -standard molecular biological techniques to construct a cDNA encoding a fusion protein part of wthich is a toxin (eg.

i maJo a jeVoa a Cui asx25 cruc Cor o~ 198, JIz1~mr~ooav~.9: 237).

Selr_- roups '_ave s'how~n rha;: an-ribodies I n.Keda zc Zoxic mol ecu as ~t ari t -L l ?co3ulICriofl exoreSSJ-Mg Cne aorre an-cecn (2-U1Z-n atal I.,e l.18) The advazltace or, jmsn7!oz~oxifl IS qa- "I aha vh are ncr- 1v seec-Jve 4 n t- a n taraet cell a* c crctvadta small doses can eii-miraz-e unwanteQ /ootent.a II rficls A var-Letv o f -cvtcjtoxin s c an be used, to zroduce Olt imfLuflocox~ls. ,Ric'in b din n Jbd- conj ugates .have been uaed to dele-e. bctL norlia -1and neoplast-ic Y cc. ynhocytsr V_-1vo and in vi tro., (Fulton, R. e t al., 198 teeN _e a 7) Qther tox ns such as *P seudomonas exocoxift A and dipoh. heia toxi have -also becojugated 'to antibodies aid usad...o sec~ nooulazlrons of cells i(Mavr, a d.Fu 1,o n R.J 1992).

In the -late 1980s and ea -lv 90s s everal. hu-man Phase T/T- *clinical trials, were performed using antibodies *conjugatedO Eo he Ricin A chain(eer et *al.1989; Sjliter, et al-, 1987; Vitetta, et ca::Oer z n r 1 w a nc eu o ource n 7Cfl ca e an- ge lt\ nn r a been a devaelojment of "second generacion" i-mrmnotoxi1: w'nicn- nave av-3.ed some of *the problemsR S at- l non-lsnec C MMt'no arenic t and toxczCty J the trea ed patients- This sznraznea u Se s' zthe Cde9avccsvlaZtedU formT o f th1e ici Chain-1 conjugated to the CuMor Soeclric antibody.

o-ne such Wha T study -use-'i chi's III.,j e FOrm~ .0 iM1"Uno0toxit- aaain-sr Call Ivo o a an 5 t atI e n. t (Vitetta, E S al., 1991). -Aceroximatelyv 40%k of. the zoatients achieved parcti-l rfemissions In. whi~ch zt-he i overall tumorl ourace--- waas reduced by S0% or more. Killing .o-f the tumor cells was rapid occurring w itchin. 1 e after completion of the therapy.

-kliof: the -n vivo stuct2.es presented herein use an, he tibod- rica. co antibodiy7dgA coalugate. T1 an n -n-jugatien was Derf ormed with a heterob-if unc-L.-ional cross-linker

SPDP

or simp-L by h e method described bv- May, R and Fulton, 992) on the OX-AO 2 r i e r, a ree amino group antibody was react with the crosslinker.and the macromolecule was ouri fied. ;The p u t i f: i ed OX-4P an ibodv.

Droduc was then reacted w4 th- reduced -ric-in A c1n a 111 e) and the hvbr-*d molecule was (whdch- has one Efree.cystein iDurified.

Initially the rat OX-40. antibody was-c'onjugated to the, darA form of -cin and tis ne~eroconiJugaze was used to 'nnoi:EnC~Z2vaitro oroljrera:ion of antigen specific CD4' T-cellI lines.. The T-cell lines used were specific oMBP and upon adoptive transfer caused EAE in naive recipients. Fic. 5 shows that-the OX-4o heteroconjugate inhibited antigen specific proliferation of. the MBP speciric T-cell line in a dose dependent manner with 501F inhibition at approximfateiy 20 ng/well. A control using Ri c in A alone showed Thnhibizion, of the assay only high concentrations 500 ngr/well and above', but no effect on the assay at, lower concentrations (data not shown).

Controls using the OX-40 antibody alone and an isotypcmatched ,unrelate6d antibody. conjugated to dgA also showed *no -inhibition (data not shown) The OX-40 antibody was then conjugated to the Pseudomonas exotoxin and this coniju~gate was.tested for Inhibitory effect on antigen: specific (MBP)' CD4* T-cell proliferation .(iues This hLet~roconjugate was approximtl 4-fold more efficient at inhibicinar the in vitro assay, and the toxin alone did-not inhibit the assay at any concentration., This assay was perf-ormed with a Lewis MBP spjecific line (Fi1g. GB) and an F1. (Lewis x Buffalo) MBP specific line (Fig. GA) with the same results,,showing that the inhibition of proliferation was not strain specific.

The OX-40-dgA was also used to inhibit'the in vitro, pro-i- ato of a CD4- T-cell line activated wizh an antigen that was irrelevant to EPAE or auto.Lrnmunlitv. The anta.iaer used was thne puriFi ed protein aerivazive antigen from Mycobacterium tuberculosis. This antigen was used to ascertain whether the OX-40 antigen was specific to CD4' T-ceils activated by the MBP antigen, or whether it is present on the surface of all antigen activated CD4' T-cells, regardless of T-cell receptor 06 rc~y As shown in -Fig. 7, there wa- a dose O dependent inhibition of proliferation of the PPD activated T-cell lifte with a comparable 50% inhibition to 0 the MBP activated lines. This shows that the immunotoxin will inhibit proliferation of any activated CD4- T-cell line regardless of the antigen specificity.

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0O4 54.

Since the OX-40 immunotoxin Was effective at inhibiting the proliferdtion of M.BP specific autoimmune CDC~ T-cells r vtro, expar-ientz were -then performed to determ_.ne tl- rotential of -Iiis immunotoxin. to. kill M4RP'specific CD4'. T-cells in vivo. To this end irradiated -rats were used initially; these animals received an ercephalitogenic dose of MBP specific Fl "T-cb~ls At the same time the animals were'injected with the A conjugate or Ricin A alone. The effect of irradiation, is to deplete the rat's immune system so that it would not recognize and'deplete the allelic variant Fl donor Tc ells. The-use of irradiated rats facilitate's the detection of the donor T-cells with the RT7.2 antibody in the host a7 the trarsfer or tnese cells and allows cre t ate o-f these t-ransrFerred cells to be determined (see wigs. 1, 2, 8A and 8B).

As shown in Table Iexperiments 1 and 2, only 1/8 animals -receiving the OX-40 immunotoxin showed clinical signs or: disease while all 8 animals that received unconjugated Ricin A ca me down with EAE. The inhibitory effect of the imrnunotoxin appeared to be mediated by the s~ OX-40 antibody since the Ricin chain alone showed the same disease, score when comnared to animals injected with erceohalitoge nic CDq' T-cells~ alone (data not shown)-

S.

4 .5 *4

I

500 4

S.

.5

S

Table I 6X-40 Ricin A Imotoxin Effect on Experimental Autoimmune Encephalomyelitis .Transfer treatment b Incidence Day of EAE *Dose'- Onset .Score"' 2 X 10 OX-40-RicIn A 1/3 6 0.66 *6 .5 X l ob Ricin A 3/3 4 6.33, Exp 2 X 10~ O0X-40- Ricin,A 5 10 X '106 Ricin A. S5 'A 9~ .0 Exp 3 X 106 OX-40-Ricin A iO X 106 No treatment 3/3 6.50 aMBP specific CD 4' T-cells were stimulated for 3 days i vitro with antigen and an .tigen presenting cells and transferred into naive recipients.

b 4 0 0 gg of OX-40-Ricil A or the same 'molar amount of Ricin A alone was injected At the same time the cells were transferred.

cValue represents the-mean cumulative. EAE score for each group. no signs; 1, limp tail; 2, hind leg weakness; 3, hind quarter paralysis; 4, moribund.

The data in Table I was generated using the EAE model where F1 M3P T-cell lines (Lewis :c Buffalo) were injected inco irradiated Lewis (parental host) recipients. Thus, the transferred T-cell population could be detected in the soinal cord of these animals with the RT7.2 (Buffalo) antibody. Figs. 8A and 8B show the outcome of the treatment as assessed by the number of donor cells isolated from the spinal cord of the treated (clinically well) and control groups (paralyzed) on the first day of disease onset. A total of 20C,000 spinal cord lymphocytes was isolated from-the control group and were found to be donor derived. In contrast 80,000 S spinal cord lymphocytes was isolated from the treated 'group and only 15% were found to be donor derived. There was over a log-fold difference in the total amount of 45 .donor derived MBP reactive CD4 T-cells isolated from the e* spinal cord (the inflammatory site) between the two S" groups (160,000 vs. 12,00). This suggested that dGA conjugate was specifically deleting the MBP-reactive CD4' T-cells in vivo.

As shown in Figure 9, animals were treated with immunotoxin on the first day of disease onset when both groups had an average clinical score of 1. The day after treatment all the control animals showed signs of complete hind limb paralysis (score while the treated animals progressed only slightly (score The mean cumulative score after treatment was 8.0 in the control group and 3.75 in the treated group.

to 0@ 0.

a a.,

S

*504 0I 5504 In a secarate experimflnflt anim.7.is w.1ere tlreated on tne first day of disease, and were sacrificed 24 hr later.

The lymphocytes wera then isolaten. from the spleens and spinal cords. Three groups were analysed.-for in vivo labelling of the OX-40 antibody. The controls received no treatment,-the second group recei ved unconjugated OXand the third group received the OX-40 immunotoxin.

Similar number of cells were recovered from the splies, but half the amount of spinal cord lymphocytes were, :J recovered from the'OX-40 immunotoxin-treated animals compared to the control and the unconjugated treated groups. in vivobinding-of ~the OX-40 a ntibodywas detected with an ant-i-mouse Ig-FITC. Approximately 15,-20%k of the lytnphocytes isolated from the spinal cords M15. of OX-40 and OX-10 immunotox-ifltreated rat Is were antimouse Jg positive. -Cell6 isolated from the spinal cords -of control 'rats were mo~use Ig negative,' even though 18'c cfz the lymphocytes. were_ OX-AG positive -(Table Te~ wera..very few anti-mouse Iqg-FITC positive cells isolated f rom the spleens :in -any of Ithe qiroups. A similar percentage .of, OX-40' cells and anti-mousb Ig' cells, were isolated from the spinal cord of the OX-40 and immunotoxin groups. .The majority of anti-mouse Ig positive del aeassociated with-the donor populati.on isolated from the spinal cord (RT7. 2/anti -mouse Ig*; Table Ia). These results demonstrate that the in vivaadministration of the OX-40,antibody resulted in the exclusive binding of autoantigen specific cells isolated ,rom Ehe inflanred cissue.

TABLE Ta: in Vivo Labelling of OX-40 Positive T Cells Treatmenta Donor'/ Cell Isolation OX-40' a-mouse Ig' Donor~ amouse Iq.

Control Soinal Cord

S

7 9 b 02 4'Ai Spleen 0. 3% 2.4% OX-40 -Spinal Cord .7.7 Sj*% 53. 2 Spleen 0.3%1, .4 46 5 'a ox-4o-Ricin

A

Spinal'Cord 81 50.3!r 18.0% Spleen 2.3k F!E T cells specific for MBP wverd transferre,_it iral te Lewis hbsts- and 400 jig~of OX-40 immunotoxin or or All t. the .pe1rcent positive' c l wr eterinined by' FACs analysis using is~type,.'control aritIodies to drawl quadrants-for negative comparisons.

CThe mean fusfllq itensity (MFI) Of- the a-mous e 1g' cell .s wa .s -ve Iry si milIar to.MF I of the OX 1-40 FITC stained 10 cells.

d The total number of spinal cord lymphocytes isolated *from the IOX-40 immunotoxin animals'was half that of-'the and t-he control groups In order to assess the efficacy of the treatment-we isolated spinal cord lymphocytes and analysed the amount *of BP reactive'donor7'cells with the RT7.2 antibody from immunotoXin treated rats versus control rats (Fl donor cells transferred into irradiated Lewis *0 reco..n) Pic 1, and 1 s'n zh ce ~OX4 ~immunczoxin adminjsterea on tesame cQO eltase as a ssessed bv the umer of vln ecie oo cells isolated from the soi_ lcord. RT7.2-oositive 5 cells were analysed the first __day off disease onset; the LreateG animal was cli:--ically well score_ 0, wit'h 1 .2 x 10" donor- spinal cord lymph,'ocy tePs) wH1Ie the control animal was paralysed (EA E score ,with 1.0 105 donor-' sninal cord lymphocytLes) In F'igs. l0C, D, E boo and Fthe treatment was given the day before disease onsetwhenhe only OX-40 positive donoTces one. Whnto el isolated from the, rats were trom the spi'nal cord Fig.

1).Spi nal 'cord and spleen--lym hocytes were isolfated 24 nr after the treatment. The number of donor-derived *eo~ spleen-cells isolated from the recipients was.unaffected by the treatment (datafnbt shown); In all the 8 9* como~arisons the number of donor" cells isolated from the ~-inai cord was decreased by over a lag in .thp -treated *-..animals (Fig.' 10) these data demonstrate that the 20C seiFclvimmunotoxin-seiial deolIeted I4BP reac'Live donor cells in vivo, and as, a consequence less. encenhalitogelic -cells were isolated f r om the spinal c or dl1 of the treate~d animals.

The same in vivo experiments were then oerformed in a non-irradiated hsaLewis MBP activated. T-cell line was transferred into a Lewis host. The non-irradiated host was used because this host has an intact immune expe rltmentZs itwas ia I' Iv csrrc a snl injection of tne conjugate at: the tim e or trans-Fer or T-cell only parciallv inhibited subsequent n-eveloumrent OE LLe a~sease. Th-erefror- exneriments, were oerrormea uLzi i7ing two injcticns at- differEnt t:I-es.

0

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SSS

1

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BBS

0~q S. 0S 0 00 "mis B B C B0~S GB B .5 Three animals were inject-ed on day 0 and day 3. and, comnared to 3control anirtials (T TlTI) .These results suggest that- the irmuaotoxia was recognizina and killing the autoimmune T-cells at-the sitea of inflammatIon on day 3 because, the only donor T-celis ex- ressing the OX-40 a-.1t igen on. d~ay 3 (the day before disease onset).

,were in the spinal cord compartmen~t (Fig 1) and the, highest percentage OfOX740 express-ion on donor T-cells in the soinal cord was the-day before disease onset (Fig.

4 0.

Table :iEi OX-40Ricin lmulrtoxin Effect on~ Exrperimfeatal1 Akutair-aune Encenhalomyelitis (in non-irradiat .ed hos t) Transfer Treatment_ Days or Da./ Of EA Dose' Tfiject~on Onset Score, 9. 0 x 106, 'OX-40-Ricin A 0/3 S. 1133 x 106 Lohn 8.25 'Lewis MBP -snecirlc CDL4 T-cells were stimulated for 3 cavs vitro with antigen ann antigen p)resenting cells 33 and transzerren _noLws recipients. -400 Ag of OX-40-Ricin A or the same molar amount atf Ricin A alone was.injected at .Lthe same time- the cells were transrerrea.

value represents the mean cumulative EAkE score for each group of 3 animals. 0,,no signs; 1, limp- tail; 2, hind ee wa k e ss; 3~ iO~ V5 me SX e, inn i-C an'-1en szec!_,ic CD4' T-ceil _unCLIoII bDoth i-2 vitro and in viva. Th e eE7e c t of th is s ne ci ric iMMUaocoXin does not seem to be rastrictad by strain or *antigen saeC"I~ nn ilms icl ave a wlae range of onlications in vo The exneriments desc-ibed above ere perforrmea usinor the r at model system and antibodies -againSt the rat- aee protein_ Howqever, the rat OX-40 anc'tibody does not recognize human or rnur-ne activated CD4' T-cell (data not

*Q

5 shown). To faciIi tate' the development of the present invention for human theraceutic -use, it is-neces sary to oroduce ant~bodies against the-human OX-40 protein.

To- that end, a human cDNA encoding the hu-man 13 otmolog. was cloned., -'Iitially, two oligonucleotide orimers were synthesized-'for use in the oolymnerase chain reaction (2CR) These primers A~ere- designed to amolifv the fEUll1 length-';Oj_ cDNA- seollence; one primer -was homoloac.u_.to_ frhe coding s tra n d. ,JLn t he region ,of: the start c-od-n ot- te ra t cDN a ir~a~ he, c ncr was the inverfse-comioleert o f the- coaig st-an7in ~t-e region of the ston codon o f the rat cOAsequence.

Surprisingly, no product-was ever robta ihed when these PCR primers were used with R.NA isolated from activated CD4* T 1 -~-'~cvtes trom S- nCe s-,ancla'rd metnhoc off cloning hUMe CD.

was unsuccess-ful, alt-e-riac-va approach was reurea.

rirst, the PCIR arimers were successfully used to clone the murine OX-40 cDNA by PCIR from RNA i solat-ed from murine CD41'P Tca= Ils activazted with Concana-v;alin A (data not s-own) Th en ne, mu O-0 cDNA a usedt too il! robe a cDN A la-mda gt-L l ib- ar-\f:r human ar:tvated"T ivm p'-hDc v "Is (Noc. H-L 03 1 pauren ased from Pec alo Alto CAM lve similar-ly sized oositi're clones (I050-1 .200 bo) were obtand- Trhase five recomwbi nant see* lambda clones were subcloned into the Bluescriot -olasmid (Stratagene, La Jolla) and- then- sea-uence-d on-the 37CA automated sequencer. (AppiD ed BiosystLemsr PasdeaC *The secuence of the human OX-40 cDNA. is set fo:rth in SEQ.

M ID No 1 in the. accompay nq sequence lIigads showfl in Fig. 11.

A omoarison o f the roredicted amino I acid sequence o f the human OX-40 protein with~r peoLtide sequences in the. Gernbank database indicated a high degree of-homology wIn the murine OX-40 and rat. OX-40 sequences;. the probabilities *that the predicted amino acid euneo the human protein shownlin Sea., No. 1 was not related to the mur-ine or rat OX-40 amino acid-sequences were predicted to be 3.4 'x l0'5and 2.9 x- 10' respectively. The'next most closely related peptide sequence gave a probability o ij.: Z!rhemr -2 177 ZCMZE.IS e~o scrnzn~ z amino acid 3, GE '"ne r ac secauence rev ealed an amfln3 acid 1 1 00Y Ot- e2.5 and a nucleotide am.mi -a acm-. stret-~ were co-served.

~ores1Onin GVHD:- 'The exi :ressiono 01-A40 onT-cells 7was. stu~ e -aient w4t reaf vr F us faost disease' kGVHD) a.

.4 *441 *4*1

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h~ree baatj4ents:_wh!o. -newniallogeneic bone marrow tr~ansplantation Came dawn with GV?'FD. The dav of GVHE) Ao onset varied between the patients t-ram 7-50 days, after transolalt. Table 3 shows. a: summar-yra oX4 expressi.on during tlhe c-VED. eoLsode- :All the natier-,Ls showed an inc reasz:d_ -0 cege o CD periphe al blood cells excnressfl-g OX-40 during t~ hearly stage of 'disease d Iev elobm en L. In all t hentinste'ecn g of OX-.0' c l s d li e a t r he initi a! clinical e is d (7-14 days PDOst 0VD -Th is -data is c(ons istent wi th theu ransient exesin of OX-4 o asee drng the e arl staaes or7diease development mnkE i.2 0-4 exrsmn-'fl GVHD correlated with the early development of the clinical signs and may have 'direct dignsiad therapeutic applications for this ah'd other human tranrsplant/autoimmufle disorders.

ox,-3 a ez Da v-s %C D 0' GO of H 2_ cra 2 .0.0 c

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aDonor and reciolent berionera b1O vta~hocv-e er' s-ene.for OX-40 axoresio orr to -m*e tr-an Isniant -orocess and. the 1, cel-l-s we're less than 1% Value 'represencs th3-e mL'aximum sevrt v each, ua L e-a obtained during clini~cal signs o f 'G Vr(si involveament). tno Signs; i greater t han no involvempent but.involving 25%s of ski'n; involvement b ut 50%- in-volvement;' 3 50-s involvement; 50% inivolvement .with:,blisters; 5 -leading to death.

CC

C Lvmphocytes from the' perinhe'ral bloodocf GVHD oatients is were isolated bV Hyoaaue- -icoll. -,Samples were isolated an nalvsd.a varipus. days p&5t L GVH. -The sa -pl s wer dual: ikai -ed. with' an an c-h-um-?i.CD4-PE anioVad an anti-human OX-40 antibodv Thie -OX -0 antibo~dy was detected wi-,th': an ant-L os TICG- -ITC. anid the. inirJ *anti-:bodV alone was used as t h neaative control.

and IH3D It has been -found th11at OX-40 c.An be of use an. ionneccion With 13SD.

-Th e tissue e xonr ess ion oF 0X40 was investigated using the standard tech-niaue of: indirect alkaline phosphatase imrunohistochemical staining (see eg immunocytochemistry: iocv t_.SSUe Sarrz:ie5 o- r e o :Do ave cazsand crohn di-se C5 aciencs sr= nej nosiz:I-elv anziOVAO an" 1odv. Clusters of oxzio0: calls weseen ang zne rrtflO1G Ch~ amn _rn:esinal Lissue from" norma=ls or in7 sanol)esz of C.l t S uin ored. .ntestnitsu-trm nyi~ tc scat~er:en 0X-' celis een EXAINPLE ONTE V I. F n.vt n ere~f oro-vlQacit-ne secruence cae numian OX 0 c~ D. cNA, one s 4 din zne art will recoan-~ _h e fu I ength cDNA Cfone c an now- readily be obtained byv standard m:ath-*noQs. -Such, nethcds include, for exam-D e, the oalvmerase chi ectn1C)by which- means. DNA *secruences can be amnlified.: Methods and condi.tions Far P9CRI aitolification of DNA are-. describedr in Trnls et -al.

(1990) and Sa.Litbrook e t a 19 1S9)1.

Thea seleczicn o~ fDCR ar-mers for ampi if icat ion or the numan. OX-40 cDN.A will be made accordiLng to the portions ote. cDNA whih a desired to be ampli-d. Primnrs may be chosen t~o a L o 1 1v small freazacots -or th e c DNA or the e nt ire cDt, mo-lecule. vat ia tan o n am cation conditions may. be re- cuired to accommitodate orimers of differing lengths; such cbnsiderati6nsara well known-in .1 .9 99 904 9 9.

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orZ examnia Cn.*te enzlire cDN,-A MO ceCU2 le to r ne n rn OX4--a -NA -mv beaoiid sn n o Iowi~ nq nPrimers a: ad 2 re also sez frt -eacmavn secue:n-ce-lsi, as SEQ- Nos. 2 a.nd r i-me r 2: 5- CACGAA CT-G-zCCACGGTGG-AGTG 3' tiem-oiate DNk -for PC.R amO!2t:catIon to -Orouce tone nurnan .OX-1 cDNA can, be extraczed frmthe law=.bda 'CT!2. c DNA libzrary f.rom Inuman ci~le 1 lvoanoc Vtes-produced by Clontecih, A, California (Ct cNo. HLIO3,!G) Alternaui- veliy, tehuman OX-40 cDNA may- he obtained: by PCR amplification o f reverse transcribed RNA (CRT-PCR) (Veres et 1-987,; Kawasak-i .t aL-,,1990) Essentialiv, total -RNA is extracted, from activiated human CD4.T-~cells- by, any o. a varet oT TMet-, r~~ useda s described' in Sambrook..et '(1-989) andAusubel et al- (1987)- Su J-table .Anumani CD4- T -,el ls inc lude- the numan CD4' T-cel ljnplhom-- tell line descie bvy cootenberg et al. (A9ltern~-atively, ac-tivated CD4' T-cells can be isolated ~om, h"uian. er-ibheral blood as described by Kruisbeek (19-)The extractedRnA is then 23 used as a temnlate o orming~ R C amplification o F the human OX-40 cD3 AL Standard methods for purification and cloning of PCR oroducts are well knZown in t'ne a:uancn a2e cescriced byi innis ez (-1990) and 'amn"0 t) r (1989) EXAMPLE TWO S With -the provision 'of the human OX-40 cDbTAk, the expression and purification of the human OX-40 protein by standard laboratory techniques is now enabled.. The .9 purified protein may be used 4for antibody production and 0 patient therapy.

9999i 999.

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.99 Partial or f ullI- iengt ,h c DNA searuences, which encode for the suhject, p rotein, may be ligated into bacterial expression vectors. Methods for ,expnressing large amounts of prot .ein, fromf a cloned gene introduced into Escherichia coli coi) may be. utilized, for the purification of the human OX-q0 protein., For, example,. fusion proteins consisting of ariLino terminal peptides encoded by a pDortion of the. Coll LacZ or LXPE, gene linked to'the rpart or- all, off the b~umant OX-40 pr otein may, be used to prepa .re polyclonal and monoclonal. antibodies that recogni ze the human OX-40 protein. Intact,. native proteins mayealso be produce d in.E- col i Jn -large amnounts for functional 'sttidies. Methods and plasmnid vectors for producing fusion proteins and intact native proteins in bacteria are described in Sambrook et (1989) 17, herein incorporated, by reference). Such fusion proteins may be made in large amounts, are easy to purify, and can be used to produce antibodies. Native proteins can be

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nroduced' i"n bactceria bv p lacing a scrornc, rec'u~laeci promoter and an efficient ribosome bidg s4t 1 sceam of the cloned g~ene. Tf low; levels of orben r produced, additional steps may be taken to increase protein production; if high levels oE protein are produced, purification is relatively easy. Suitable *-methods are presented in Sam~brook et al. anad are sC well known in the art. often, proteins expressed at high ~levels are found in- insolubla inclusion bodies. Methods, '5 for extracting proteins from these aggregates are.

described by Sambrook et al. (1989) (ch. 17) Vector -systems suitable for the expression of IacZ fusion genes a. include the pUR series of vectors. (Ruther and Muller- -Hill, 1983), pEX-l-3 Stanley and Luzio, 15,.84) and pMR100 (Gray et 1982). Vectors suitable for the production CC of intact native proteins include pKC3O (Shimatake and cC Rosenberg, 1981), P117 7 3 (Amann and Brosius, 1985) and pET-? C tudiar a I d. Mof fatt, -198 G) .Hu man OX-40 fusion ioroteins may be i~zolated from 'protein gels, lyophilized, ground into, a powder -and used. as-.antigen preparations.

For expression in mammalian cells, the cDNTA sequence may be ligated to heterologous promoters, such as the simian viLrus (SV)40 promoter in the pSV2 vector (Mulligan and 2S Berg, 1981), and introduced into cells, such as monkey COS-l cells (Gluzman, 1981.), to achieve transient or long-term expression. The cDNA sequence (or portions derived from it) or a mini gene (a cDVTA with an intron and its own o:romoter) Is introduced intc e~av i exzDression v=-ctorsbv conventional zcniues. These vectors are designed to permit the. transcription of the cmmk eukarvotic cells by providing reizula-tory secouences that initiate and enhance the transcriation of the cDNA..

Vectors containing the promoter and enhance-, regions of too..: the SV40 or long terminal repeat (LTR} of the Rous Sarcoma virus and volyadenylatiof and. splicing signdl f rom SV40 are readily available (Mulligan et al. ,1981; G-orman et al.,,:1982). The level of expression of the cbNAk can be manipulated with this type of vector, either by using promoters that have different activities (for .9 example, the baculovirus pAC373 can express cDNAs at high *levels in S. ffrugperda cells (Summers and.Smith,. 1985)) OV i%*5 or by using vectors that containpromoters amenable to modulation, for example, the glucocorticoid-responsiVe p romoter from the mouse mammary tumor virus (Lee et al., 1982)_ In addition, somhe vectors co ntain, selectable markers such as the crt. (Mulligan and Berg,, 1981) or, neo (Southernand, Berg, 1982) bacterial grenes. TheEse selectable markers permit selection of transfected cells that''exhibit stable, long-term expression of-the vectors (and therefore, the cDNA),. The vectors can be maintained in the cells as episomtal, freely replicating entities by using regulatory e lements of viruses such as papilloma (Sarver et al.,'1981) or Epstein-Barr.CSugdel et. al., 'e lraJC~ one can as'o± nes that navre lnzegraued che Veczo in r eomic DNA. 3oth ofI these tVOCeS o: CEll lines oroduce tln gene product on a continuous basis. one can also produce cell lines thiat have amplified the number of copies of: zhe vector (and therefore of the cD-NA as well) to create cell lines that can Qroduc hih levels ofr-the gene product (Alt et al., 1978).

4 atI ease

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J3The tranrsfer of DNA into eukarvotic, in oartiJcular human or other mammalian cells, is now a conventional technique. 'The vectors are introduced into the recipient 4 cells as Dure DINA .(tranrsfeccioi) by, for example, precipitation with calcium phosphate (Graham and Vander O Eb, 1973) or strontium phosphate.-(Brash et al., 1987), electroporatiol -(Neumann et 1982), lipofection (Feigner et al., 1987), DEAE dextran-(McCuthan et al., 1968), microinjection (Mueller. et -1978), prot6plast fusion (Schafner, lSSO), or pell et guns (Klein et al., 1987) Alternatively, the-cDNA can be introduced by infection with virus vectdrs. Systems are developed that use, forexample, i.etroviruSes (Bernstein eL 1985), adeno .viruses (Ahmad, et al., 1986), or Herpes virus (Sate et 1982).

The human OX-40 protein-expressed in eukaryatic cells may be purified and used to produce antibodies. The human O-0protein may be extracted following release of the tesuoernazanz or, S eallerce ma'.' be inCor0oraC=ed 1into a eukarVOtic e:csiovectLor and, expressed as a chimeric orotein with, for examole g-globin. Antibody to 9-globin is th-ereafter used to our ify the chimeric protein. Corresponding protease cleavage sites engineered between the .8-globin gene and the cDNA are then used to separate the two polypeptide *-fragments -from one another afiter translation. One use'ful expression vector for generating j3-globin chimeric proteins is-pSGS (Strataana, La JolCA) .This vector encodes .rabbit fl-globih.. 9.

6* 4 006 0e 9

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4.

This invention encompasses recombinant cloning vectors containing the human OX-40, cDNA.seo'-ience, or portionsthereo f The human OX-40, cDNA is operatively linked in the vector to an expression control sequence in the: recombinant DNA-molecule-, so -that- the human polypeptide, or a portion thereof, con be expressed. The expr essiol control sequence may,,be selected-from the 2 0 group contiiisting of sequences that control the expressioa of genes of prokaryotic or eukaryotic cells and their viruses and combiniations.-thereof. The expression control sequence may be specifically selected from the group consisting of the lac system, the -trzp syscem,- the tac system, 'the trc system,, major operator and promoter regions of phage lambda, the control region of ifd. coatprotein, the early and late promoters of SV4O,,promoters derived from polyoma, adenovirus, retrovirus, baculoviru-s and si.mian virus, th oomczer r3ocnolcrt kinase, the nromotcrs of yeast acid. onosonamase, te 0ooot- or. tne yeast alnha-mrat'ing f-aczors and combinationls thereof.

The host cell, which may be transfected with the vector Note*: o 7 this invention, may be selecte d from the group 00.4 con'sisting of E. coli, Pseudonas, gacillus subtilis, Bailu~tarter 'hlus c~r other bacilli; other.

bacteria; yeast; fungi; i.:nsect;, mouse or other tissue cells, including huiman tissue culture cells.

in a preferred ernbodimeht o the present invention, the full length human OX-40 cDLNA as' shown in Fig. 9 (from start codon to stop codon) is; ligated into a baculovirus vector- and the recombinant hiuman protein-is 6rbduced in -:'the atppropriate3.nsect cells,. Suitable baculovirus excpress2io systems~include Ehe B 'cPAK.T' Baculoviru *Expression Systpm produced by Clontech :(Palo A lto, CA) Thus, by way of example, the -full length-human OX-40 cONA is ligated into the plasrnid pBacPXKl and expressed in *Spodoptpra, fugipez.da cells according to the manufacturer's-instructions.

The human OX-40 protein produced in the, insect cells is .hen-purified by standard techniques. A preferred techniqueof isolating the recombinant product is to use *a vector that adds an additional 6 residues of histidine ta the r-ecomhi-ant oroztein. F= o=.eis n-oauced this mrnEr.11- che-at etl f2C ac.tasarti zouri i'_cation enormously Thus, f-or example, In high sat olyhi scidie us o roteins b~nd wit-h a high affinity to a metal chelate matrix.whereas the majority of hos t proteins do riot bind at 'all. Low afinity a~*O 1 16 inding host proteins can be washed off the matrix by decreasing the 'pH to 6. 0. Specii elution at the Doiyhisti4~-Jne.onftailifg fusion protein cai! be.

acco molished wit.h 3 00 ml imidazole buf-fer at oHr G.0.

EXAMPLE THREE M ronoclonal antibodies may be'produced to the human *protein for therapeutic use. Substantially pure human S OX-40 protein suitable for use as an immunogen is 0 isolated from the transfected or transformed tells as described in Example 2 above. Concentration of protein n the final preparation. is adjusted, forexample, by *concentraticn on an Am Loon fIlter device,. to the level of a few milligrams p er mill/ilitre., Monoclonal ~antibody to the protein can then be prepared as follows.: A. Monoclonal Antibody Production by Hybridoma Fusion.

25 Monoclonal antibody to epitopes of the human protein identified and isolated as described can be *prepared from murine hybridomas--according to the classical method of Kohler and Milstein (1975) or ~w.tna ew -2lcroci-ms a- rn hemouse is tzhen sacrificed, and t~he antibcdy.-producing cells of t he soleen isolated. The spleen cells a re 'fused by means of polyethylene glycol with mouse mveloma cells, V* ~and t'ne ex.1cess unfused cells destroyed by growt h of the *system on* selective media compri sing amincoterin (EAT B eda -Th ucesfll used cells are dilutd and a1icumo ts ofr tne dilution placed i n wells of amiicrotiter o late where growth of the culture is ccntinued.

Anst ibodv- oroducing clones are identified by detection of antibody in the supernatant fluid of th Ie wells by im~munoassay procedures., such as ELISA, as originally ~fsdescribed by Engvall (1980)., and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody~product harvested for use.

Detailed Lrocedures for monoclonal. antibordy production a r described in Harlow. -and Line (19188)_. B. Antibodies Raised .Against Sy-rthetic Peptides.

a alternative anprbach to rai-sing antibodies ,ag4 Jn s the hua X40poen is to use synthetic- peotides synthesized on a commercially ava.ilable.-peotide 25 synthesizer based upon the p r e d3 6ted amino acid ~seauence *of the human OX-40 protein shown in Figure_.'11.

In a preferr ed embodiment of the present invention, 4iJ eq..

C.

a C a C S

CC..

2 oroLe~r are z~oduzced~ Onr imaliv monocl oral antibodieS raisZad aaainsute "man O:(-40 no~~_el.C~' de!tect the human Q.X-40 protein. That: Is, such antibodies_ -ecoanize and :bind the human OX-40 or-otein and do no t substantially recognize or- bind to other proteins found hn uman cells. Put another wav, such antibodies have a specifici-:ty of binding n numans tcsubstantially only the -uman.OX.-4O proteir and-- thus t~substantially only activated CDA' T-cells.

The determination that an antLibody specifically detects the human OX-40 oratein is made ov anv one of a: number of standard immunoassay methods; for in-stanc6, the Western blottiig-technique (Sambrook et al., 19989). To d e terffi:inh that a given antibody preparation (such as one produced in a mouse) specificallv~detectsthe human- OX-40 pro-tein' by western blotting, total cellular protlein, is extracted from h1;manc e IIs that do not _exps th6 OX-40 antigen, 0 such as non-activated lymphocytes. As a positive control, tot l cellular prti ~as xrce from activated T-calls especially for e .xamnle activated CD4*T cells. These protein prepar ,ations are then electro'horesed on a sodium dodecyl sulfate- 5 polyacrylamfide gel. Thereafter, the proteins are transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane. After washing the membrane 2 *sees zo remove ncn-spcC iZI 1 i.es thelreenc or se~i~ICI~iV 0 -C he use oan anti-mouse an'tibody conjugated to an enzymne such as alkaline Dhosnhartase; application off rthe substrate br omo-4--chl-oro-3-in-,dol-vl iohosohace/nitro blue tetrazoiuM results in the oroduction of a dense blue comnound by mmuno-loCaliz _ed alkaline ohosohatase Anioiswhich soecificallv detect the human OX-40 proctein will1, bv this t echnicrue, be sho'qi' tc: bind to the hum~an OX..dO Protein~ band (Which will be localized at a given uoslti.on on the gel determined by its molecular weight). Non-specifi-c bindina of the atbovt-he reLsmay occur and may-be deatectable as a weak signal on the Western blot.

Thenon-specific nature'of this-binding will be recognized by one skilled in the art-by the weak signal obtained on the Western. blot relative to the strong primary signal arising -From the _specific antibody-human OX4 rtein, binding. Pre ferably, no aatibod wqo'ld be round to, bind. to nrote-ins--ext-acted from t~le unactivated T-cells esuecially for example.CD4'.T-cells.

in addi tIi on to b i nd i ng 'assays using extracted proteins.

monoclonal antibodies raised against the human orotein are tested to confi rm their -ability to recognize th prpit eltype (activated human T-cells especially for example activated CD4' T-cells) by conjugating the human OX-40 antibody to a fluorescent tag (such as FITC) and analyzing cell populations by FACS as Oreel-b-l recoanize accz.vsiec :-cells eS:Dc1;iSI±VZ act",ra le CD a-TC "1 fce dual a n a Of an MaC~tivaced T-cell -ac-o uIa tI'o n .,ith C-D-PE and S FITC sHould show cells tla ar double csitive.

Monoclonal ant-ibodies for Use in tthe zD-eSe- o genearally be of tE 1gM or IgcG isotypoel and wii l zeferabiv be of mouse, numan oricher :!,amral an oriJr.

S

in one. Dreferred ,embodIment of the, oresen: netin h ronoclonal antibodies tLhat recounmiZe the human antigen are mouse monoclonal antibodies that: have been "humani zed". Such humanized antibodlies can be more safely administered to human patients than c an unmodified monoclonal antibodies prod4uced, in mouse cells.

Monoclonal. antibodies produced in non-human cells, -such as mouse cells, ge qerally evoke an. immun~e response when administerFed tz a humnan host, thus limiting-the duration or the biological efficacy of the monoclonal antibody (tsee generally, tJ.S,. Patent No. 4,731,244 and WO 89/06976). Humanized antibodies -are produced by recombinant bDN technology and generally comprise the antibody constant region from human monoclonal! antibodies combined with the variable (antigen recognition) region from the mouse monoclonal 'antibody that recognizes the target antigen (in this case the human OX-40 protein).

Because only the variable region is of mirine.origil, 6 2 7.umanzed zmonoccnra_ are V o dacea 2' Llmue r-esponse wnen ad- s e rec 0 a numan oet SMethlods for humanizing -antibodiJes are described by' RiechmannD et (1935), Riechmann et al. intrduced the si-..X raial regions frm~eheaxy and 1 1gh cha.n.

domains of a rat antibody into a human igGI aacibowydirected again--st human i-mphocvtes. Riec'amann .e:al.

showed th.-at thi 1:s "humanized" antkibody was able to bind 'to i ts taraen antiae n vivo wiuhnout elicininu an ant *immunoglobulin immune response.

or the preferred embodiments of this invenrtion, intact I monoclonal antib,'odies are used. However, one skilled in the art will recognize -that portion.§ oE monoclonal -ee. antibodies -that are-capable of recognizing and binding t o ago**: hz~ human 1)X -Q p oroIt e in may al.1so be employed. These a .1nihody vfrarients 4aenerally include Fab, Ftab) -~and Rr~ F ragments of antibodies whi ch 'recognize the humian pnooteix.' ImrunotO-Aclns .cormorising antibody Ifrag-me nt s have been shown to be effective i n delet inco CD4- T-cell2s both in vi vo and in vi tro using an anit iody thac recognizes all cells th at e xzor e ss the CD4 antigen CS tr ae et al., 2 E5 1987) EXAMPLE FOUR *As set forth in the preceding examples, this invention enables the production of monoclonal antibodies that, in :-ui e- as discussed below. IVhC ~ae r cmol re erred toC -mmunotz-fl5.. T±mmun-to xlls are characterized byv two co1M0Cnent S; ac:Otox 3-c nt whi tnch.

s usualliv f-atal cto a c e~l vn ttaoec or absorbed-. and a 'del'Iverv veznhclel wi serves z-o deiiJv-: the cvzou~x--P -0,e zlz _=ce'Cl tne O~asent CC nVent.:on, targe: t c I tvoe is activated T-cells esueciallv for examole activat.e T-cells and tl,%jdelive-rVvenic± e s a" anzibody or antibOody fragment t hat ecogni zes -nd bin:dst-o z the human.,OX-401, ant igen, as described in Examole 8 b elow.

*A nuimer of sui'table cytotoxins are, known in the art:, including:. cytotoxic pro .teins such as the Ricin A chain, tne deqlycsylatei 'orm of the Ricin. A cha i (dri the Pc~eudornonas exotox i n; 1.radionuclid--_s'_ suc as iodine-131, adriamyci.n and c is -plat inun;- (See. generally, Olsr'es and Phil (19S2), and Baldwin and Byers (1985)). Tn one embodimeent of the present inventiion, the c-vtotoxil iS the delvos~a~d~om t neR~~nA chain, as described i n tLS_ Patent No0. 4,590q, 071 A selected cytotoxin may be. conjugated with an antic-vtccox-s cv z nuae f e±,ccrs*a ceer.ijv descr bed,-in Tino ce er- a: For xam z)Ie, wreean cvtotc-Oxc ace- IS cDroce-nri s-uch as Z:ne Ricin A chain)~ a ~v is a7- inc,-aczc Monoclonal <arntbod-,- z--e may by way or ne~erzzunc~.%-rscs schi as carcoc'&* glur-eralehvi 1r-ere m.C:c 0 rOQUiTI imUno.'din Usn Zie euvosylz R cn chain a re a.e Rb t-nt -No& 4,590,Q7', and 7 D 89/06967, wicm are he-el n i-'cp -ai b raEerence.

iURn (D oto0Xris as po,,vided by the r as en z invention aridr-oduced as destribed aL-we are subseqruently tested to co** a c in-m their vi tro ef-fic-acy. Suchin~ vf cro testing is Deirformed using human CD4- T--cellls and the methods dezscribed above. For examp Ic an i m a iu r o t o x produced accordingr LO the, or&-ant'inventi an (e:a cvtotoxtfLconjugated to a :4monoclonai -ant ib1Po dy, t hac has been shown.

*to be speci c-o -heua OX4 rtaL. is tested us ing izi viLro ni bit i on studies-on MBP' s b acai c CD4'T cl ines -From mu Ct b sclerosi "s;,tens ~mmunoLoxins piotentially suitabl- fo s nnmarthei:,ra--v are those capDable of inhibiting -the in vi tro proliferation ofl such cells.

Since these immunoto~cins are capable of inhibiting the in i 7 vi tro ora ~oifr~r a:.ono a cniv a zO CD-' -cecls. ffrom MuirOle sclercsis natients, they should be capaleo inhibiting :hz oroliferation qof a 1 1 activated CD4_ cells, regardless of origin.. This conclusion is supported by the evidence set forth above, wherei the ra: immunotoxlin was shown to be effective against MP9 activated rat CD4' T-cells and PD activated rat CD4' T-cells. To confirm that the human OX-40 immunotoxin has *-this gene'ral activitV01, similar in vitro poroliferartion -'are q studies as described abcvz! may also be Der-ome wth human CD4' T-celis specific for other antigens (such as heroes simzlex virus.).

In an alternative embodimnent of the present invention described, n Example 5,below, anti-human OX-40 antibodies -can also be used to diagnose, cond.itions mediated by activated T-cells especialliy for example activated CD4' Tcells. Fc., such applications, it -is preferable that the 6. anntiLb dy is cconjugated_ to, a -suitable- chm~a tag'. which fLacilitates detection of-the antibody. 'Suitable moliacules include the fluorescEnt molecules fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) as utilized in the present invention.; EXAMPLE FIVE In one embodiment of the present invention, monoclonal antibodies that specifically bind the human OX-40 protein are used to detect conditions mediat ed by activated Tcells esoeciallv for example activated CD4' T-cells. For such purooses, human OX-40 antibodies are conjugated with other molecules; such as fluorescent markers.

Biopsy samples are taken from inflamed tissue for analysis. One skilled in the art will recognize that the source of the biopsy sample will vary between different conditions. In the case of multiple sclerosis the lymphocytes will be isolated from the CSE, while in oQ. rheumatoid arthritis the lymphocytes will be isolated 4ea from the synovial fluid of inflamed joints. In the case of transplant rejection biopsies will be taken directly from the target organ during a rejection episode.

*i In a preferred embodiment, a biopsy sample taken from a patient will be fractioned into a lymphocyte fraction (by methods described earlier; see Materials and Methods) -The purified lymphocytes wi11 be stained with the FFLTC antibody arnd the percentage of positiv; lymphocytes Thwill be quantitatedon a FACScan -apparatus.. This percentage will be' compared with the percentage found in healthy individuals. Any statistically significant' increase will provide an early indication'of an inflammatory event and will lead to early diagnosis of autoimmune disorders.

EXAMPLE SIX orthneraneuLtic applicati.ons, such as iireartment aautornmufe inflammationls associated with multinle sclerosis, it is anticipated that the presence of S activated T-cells especilly For example activated CD4- 'Icells at the site of inflammation will be established before treatment is commenced. The presence of these cells can be established u-sing the diagnostic methods describ ed in Exampl aoe- If the diagnostic test CPe5ao oroduces a result indicating the presence of activated TsoC S cells especially for- example activated CD4- T-cells at thle inflammatory site, then therapeutic applicaction of the' immunotoxin may be, appropriate.

For the-rapeutic adminitaonfth imm~unotoxins for C treatmenit- of conditions -mediated by activated, T-cells, especially f or- example ac tivated, CD4' T-cells, standard bs*:.

published protocols that set- f rt.11 trFeatmrient, regime-s usi4ng ,imunotoxins Mal, ut'ilized. Th'ese in~j ude, protocols described by Vit ettal et 1991, arid in W089/06907. ThlesedounS are herein incorporated by r eference.

in general, the method of treating a. patient suffering 25 'from a condition mediated by antigen-activated T-cells, especially for Ekample activated CD4 ,T-cells will comprise administering to the patient an effective amountL of an antibody (or a portion of an antibody) conjugated 68 with a cvtoloxic agent wherein the antibody (or the portio'n o f the antibody) recognizes and binds to the human OX-qG antig en. As discussed above, antibodies and -portions of antibodies conjugated with a cytotoxic agent are commonly referred to as immunotoxins. Effective amounts o~f these immunotoxins may generally be referred to as a suitable dose of an immuflotoxlfl.

a a a. a S. *a a a

S

-a a..

a. -a US a

C

a *Iaa .5 20 one skille-Ji in the art wi.Il1 recognize that any dose 6ff the imm-notoxins grea ter than zero will have some effect on the activated CD4' 7-cell population in a patient.

However, siuitable .doses are limited by the onset of adverse sideleffects oil high doses of immunotoxin.' As described in W89/06967, for imrunotoxin's comprising a monoclonal antibody conjugated with. the ricin:A chain, suitable doses. ar~e in the range of 1.0 mg/kg daily for up to 14 days., As described by Vitetta et al.

(19911-, for irrmun:r oxifls comprising antibody fragments :chast± F'ab' f ra vment~ l ,ink-ed: to 6the chemically deglycosylated ricin A chain, ,doses'will preferably be in the ran ge of_25 150 mg/n 2 -EXAMPLE SEVEN.

one embodiment of the present invent ion is :a kit containing monoclonal antibodies, tha.It recognize the human antigen. Such, a kit-would comprise a container within which the monoclonal antibody is contained..

n-f ofe emocclmen r 0 suco a tc~, e wou _Ic conrtauthe roocoaanioyin a Zormn Conjugate'd with a cvt~ozo-cjn, such as dgA, whenrby the kitz could be used to treat oatients surrering from a condition mediated by S -activated T-cells especially for example activated CD27 *T-cells. This antibody-cytot-oxin conjugate would preferably be provided in a form suitable for administration to a natient by injection. Thus, the kit might .contain the anrtibocI-v,-cytctc:Lto cornjugate in a suspended to-ino form, such as suspended in a suitact-e Dharmaceutical exciojent. Alentiey th ojgte could be in a V solid form suitable for reconstitution..

S *S S S~B bE

S

5. 5 5 65

S

SS .5 In an alternative embodiment, the kit would contain the monoclonal antibody in a form,,suitable -for ,diagnostic use, such as conjugated to a fluorescent: -marker. Such kits would be used in the detection of inflammatory cund i.Lons medi .ated by activated T-cells as-,pcially for excu~ule activa' ed CD4* T-ceils..- The foregoing eaxamples are illust-rative of the present invention, but are not limiting. 'Numerous variations and modifications on the invention as set forth can be effected without departing from the spirit and scope of.

the present inv~ention.

0 *8038*

S

*s0.

S

ala, 89 EQ ~a a S a BaSS,.

0

SOS

o 65 -S S ma S EQ 5555 o

SO

0@500I 0

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,0a~.

Abuad at al.. (1986). .7 .~2 AUann and Brosius (1985). q~~n jQs193.

Alt et al. (1978). T7. gjol., Cha. 2M:1357.

Baldwin and Byers (Edo.) (1985). *Monoclonal Antibodies far 'Cancer Detection and Therapy." pp. 1.59- 179,; 224-266.

Bernstein et al. (1985).. !Jan. 90=1SZ.235.

Birkeland, 14.L., and R.N. Barclay (1992). Abstract: 15 Bt~nteratiolal Conciress oUft.DmB~p1logy W-16(11~:82.

Bourdette,. D.N. at al. (1991). J. Neurplai. Ras.

20 Brash at al.z (1987). V_01, C611L.a.ID- 7_2013.

Casp.E R.R. at al.. (1985). T1 of IjMunl 25 Cobbold, .P etelI. (1964).L Ra=i =a:548-552.

C' i sh, J3 and Lipsky, (1988). &tAitnflan Eihumi9 31(201:1230-1238.

30. Enqjval 1 (1980).. EzDml,7.Q: 4 1 9 Feigner et. all. '(1987) Prop., NtI, Mad. SL~ 84.7413 35 Fultan,, R..e t: 61. (1988). CPaihee! Ragiearch% _4:2626-2631, Gootenberg a'E~t al. (91.~~ML Z~Dari!efltal Mdicine, 154: 1403-1418.

Graham and vander Eb (1973). Vio~ j4 6 6.

4S Gray at al. (1982). Progc. NatL,-Ad, Rj IA Gorman at al. (1982),. ZroC. NaI. Aroad. ci UFA 21:6777-6781.

so Harlow and Lane (1988). Antibodie8. -A tberntkn Hanal 1 Cold Spring Harbor Laboratory, New York, -71 Innis at al.. (Edo.) (1990). PCR PFc0tocals.- kCuide &g Method5 A nd Api1ictiOfl6a Academxic Press, Inc,, San Diego, California.

K~lein et al. (1987). Naur 27:70.

Kohler and Hilstein (1975). No~M 256;495.

Kawasaki at al;, (1990). in PC& ProocolS._ k guaide to Hathods and AnIi-gtio s, Innis et 21-27, Academic Press, Inc, Sanl Dieqo, California.

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D.H.

ge 15 HarguliesE.4 Shavach, and W. Strober, eds.) pp.

Greenet ublish~ing-id Wiley-Iriterscience, *9**gNew Yori.

:Lac at al. (1902). flaturn 204-229.

Malletti s. et Eim 9(4);1063-1068.

0 May, RAD., and Fulton, .(1992) In Vitro MathodAsof Toxioolbqy.(R.R. Watson,.editor) pp. 9-20.

*is: 25 CRC Press Inc., Boca Raton, Florida.

McCuttlart et al.: (1968)., 3. Hjt1 Cnier Irnt.

41:351.

4 0 30 Mueller at al.. (19.70). a 1 1. 5 79.

Mulligan and Berg (1981).- P roc Natl, A cad_ 1~A Ml:2072-2076.

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Sarver 01 eal (10) l.call BL 1:486 72 schafnier (1980]. Prc ~I cd gusk 22: 2163-2167.- Shizatake and Rosenberg (1981). Ntmrs Souxthern. and Berg (1982). JH Ml, AVOI Gen~et, 1:327-341.

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-Stairman, L. (1993). icentific JAnerican September: 107-14 Street, N.E. at al. (1987). a. of 1x~unel.

Mt1jz734-.1738.

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168-190.

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Weiner at al. (198). cancer isesearch A: 406~2- 4067.

-73- Ci) (ii) (.)ENERAL

INFOPYMATMON

APPLICANITS-- An~drew D- Wan- l TLEOF IkVVI1N: TRApIT OF CD4 T-CL NMIATM)

CNITION~S

NUMBER OF :SEQUNCES:. 3 CORRE~pONDENCE

ADDR.ESS

1 DDREKE Ri~hard J- FolleYr Raq K~j~nquist spaxkmafl Campbell Laigh &i WhinStOn see Ve (iii) (iv)

*.V

I: (B sTRET; 23- w.,salmon street, suite 1600 cxTY: Portland STATE; Orsou Co0UNTR. Unitend States of Amuerica ZIP: 972Z04.

KDIUMVTTPZ DisZ, 3-V 2 1i1c= z C MUTR. IBm Pc- compartible OPKR&T2.KG 5Y&TEI2 M6 DOS SCID) 50!EAWL -wordPaef eat,50*1 (vi) WiRENT MPLTTI DATAr 1.PPICRT1014

MUM:

FZLIEG

DATE:

cc) ~smktN 010) 1t0 AFFLICAtION 3A:' NIA AP0PLICATION

NUMBER:

FILING DATEr (viii) ATTOPJM/AGM'

INORMTION

IWEE Richard T. Polley, Esq.

()RZGi&Tiot1Q NUMBER. 28,107 REM CE06 NMER: 4282-35549 (ix) TELE CONNICATION IMI

TION:

TELEPHOKE1 (503) 226-7391 ELEAX:(503) '229-9446 74 INFORMATION FOR SEQ ID NO;1 SEQUENCE CHARACTEJ&ISTICSt LENGTH 848 barSe pairs ()TYPE: NUCleic awid GT3FJDZDMS Dob TOPOLDGY.i Linn=r (ii) pOLECULE, TYPE. CD1NA to WINA (ii) HYPOTHETICAL, Hafc ANTI-GEHS~t No v) FPAG!DNT TYPE.

*(3ti) 9LPQUBNCE DEScRnyrION: SEQ ID NO: 1 .jcow AGaBhaOG 14 'AM TGC T GTG G=T00 CO~C 0 QM~ CC O? @0 (M T 59 "t cyn Val gly RIB erg arv lsa 9gy eGv 9 1 T pro CY* ala. dC CCT MeO 1CM CCC Lam CT AOC &CC~ a=O ECO OW CheCA 104 0*Lu Lou LIU gly gLy lon. &Or t)ir Volt~ l o hi.

20 2 in1 Tor M! U=0 MA C CW A= MC "cOM T T= en C O. 149 ~~ae Val. aly a"p thr tyr V=o bar, am *UP yeeEhi 31 40,4 TOO C cOA Wf ATC OW A=C 0CW 7W WC WOC T=O aR9 IL 3 ae eg pa gy mn g.y al dii sarg erd ar erg ar 91n 31C oT TO 02 OG OO. TrC Tw A= fac am 23 an tbi, Val cyr r E ays IRLY 91?q vrpb tyr &an amrp e 20 65- 70 7 GTC M &A l 0 ICA O A010TTmC T B v.1 Oa- mur Sym Pro ays lye pnM aye tiir trp ayC" A1 1 dcG POT 4RA 00 AMCAG CTA TGC ACG GCC ACA C&h G&O Aa 329 wargl ea gu rg yEqln5-u yv thr els thr gO.o sop thr *95 10010 GTC TOT c00 TOO 00M 00- 000 ACC C&G TOO 01 A 30YC 3 7 Val ca erg aye amg gly thr gl" sec, Lou ap bar~ ty lye 110 .115 120 CCT OGTT CACO TOT 000 CCC ICC OT,.Cak GGG C&C TIc 1=CC 419 as pro qlif VAL amp cy. a1e pro Gyp pro proagly hi.s phe a= PCP 12!2301 135 800 a GAAC CMa G= TOO ARC CCC TG hOC AM0 TOT Ac IO OTT 4"4 gly asp &on gin _eye lye pro, trp thX thr CYA threu UVAl 140 ,G A CAC =C M1 CAB CWOOCC ACT UT!PAC TCG GA CA ATO 509 iyye liLe thr IOU gin pro 81a ear amn @Qc ec a@P S-I 15! 16015 75 be..

CC CC C C C be.

TGT G" Afi C A=C CAtC CCC CCtA CCC KCG ChG CCC GAG eye gin asp axg asp pro pro ala thr qjn pro qin 170 175 cC~ CMZ a= AGO =0 A!V A=c GTC CAC OW AMT gly DEC P=e Gaa arg Pro ii.t tta .1 alin pro thr 185 t C=C AG1R A=C TCA ChG WA C= J= ACC.CrA TCC CG 10 ywo arM thr nor gin Sly 9=o ca thr searxa 20020 am W C= AYT weC G= ]L CmV Qa CIV W-K gly gly ixg ale Val cla Ala ile IU gly imu gly is 215 220 SAG C W CM CCA C=C CMC ORO I=C CMV CMGM CCC glyIm OU lyPro lez map 11 16a Lu &laI&.,u gly3.e~ ina.g 2303 =g 'arg asp gin erg Ivu pco pro asp hUs Lys 245 250 25- 9M GCT C= IC I CM CM C giyr glyw s h* pkaerg %hc pro Li., gin i gin g;in 1 30 his. ow tbxm lse val Lyn Vb 275 GAG ACC GAO gila thr gin mI C=C IV glu trp 195 GM GTC Coe gin P"o 210, CTQTG OT I IOU Val IOU 225 TAC CTO A=C tyr Ian LIS 240 cc cur a pro ro 9-y 255 G= GAC oCC ala, "D LAL 2.7 a *O CC C C CC

CCC.

C. C CCC Ce

C

C C

CCC.

be 76 0 t 0 *fl.~ 0.

S

eOfl.~

'S

0*te 0 *8 *O

S.

S. S S S. -S INFORMATION FOR SEQ I.D NO:-2 SzQuENcs mmARAERISTICS.

LENGTH: 24 bawe pairs TYPE., Nucleic acid STRANDEIESS. SingL8 (ii) EI'O~fl~L~No, (iV) AI-SENSE:

NO

(NJ) SEQUENCE DESCRXWYZDN SEM ID NO: 2 LD ATGTGCGTGG GGGCTCGGGC

GGCTG

-INTMGMTION TOR SEQ ID NO: 3 SEQU321ZE CaRA~CTMUTIS LEN=.'h U 4base pairs (I v'pK±- Nuclic aauA STA ~ZS SluqI* TVOWGYd Linear.

(XL) SEQUENCE DZSCU71'okt -Sn ID ND: 3: a. 'Sq S 5 *5S

SSS*

Claims (30)

1. A nucleic acid having the sequence shown in Seq ID No1 or a sub-sequence of 82Q ID Uo I which encodes an antigenic pojlypeptide; or a variant or allele, thereof;. or, a complementary strand thereto.

2. A nucleic acid according to claim I comprising nucleotide.bases 15 to 848. shown in Seq ID No I or its :complementary strand.

3. A polypeptide. comprising an amino acid sequence encoded by a nucleic acid according to claim 1 or claim 2, or a derivative thereof..

4. A polypeptide according to claim.3 which comprises an amino acid sequence encoded by a sub-sequence-of the sequence shown in SPQ'ID 14o-1 and 'which includes an ant igenic determinant. %so. 20 5. Aspecific binding agent which can'specifically bind to a polypeptide according to -claimi 3 or claim 4 other than a protein consisting of the full sequence of human gp34 protein as defined in S.,Miura et: al. M61. Cell Bi1ol- 11(3) (1991) pp 1313-1325.

6. A specific binding agent according to claim 5 which specifically binds to human Ox-40 of amino acid sequence encoded by the coding region of nucleic acid sequence Seq ID No'1, wban said Ox-40 is present on-the surface of _activatedI' T-cells._ *4 qe S S 0S4 I SS* S SSt~

7. A snecif ic bi-ndino agent which can stoecif ically birid to a polypeptide according to claim 3 or 4 and which comprises an antibody binding domain.

8. A specific binding agent according to any one of claim to 7 which comprises a monoclonal antibody or a binding fragment thereof.

9. A specific binding agent according to claim 8 which is *:1O at least vartially humaiiised. A specific binding agent according to claim 9 which comp~rises a humanised monoclonal antibody. *15 11. A specific binding agent according to any one of claims 5- to 10 which further comprises a molecularly linked ytotoxin.

12. A specific binding agent according to claim 11 which comprises a Fab, F(ab') 2 or Fv fragment of a mnonocl~onal antibody conjugated with a cytotoxic agent.

13. A specific binding agent according to any one of claims to 12 which further comprises a label.

14- A specific binding agent according to claim 13 wherein the label is a radioactive label. A apecific binding agent according to. claim 13 whirh 7k) comprises an antibody conjugated o a fluorescent label.

16. A nucleic acid sequence encoding the amino acid sequence of a specific binding agent according to any one S of claims 5 Lo

17. A reolicable expression vector including a nucleic acid sequence according to any one of claims 1, 2 or 16. I) 18. An prokar-yotic or eukaryotic hos5t organism which has G.V. been transformed with a vector according to claim 12.

19. A pharmaceutical composition comprising a specific binding agent according to any one of claime 5 to 14 In 15 combination with a pharmaceutically acceptable carrier.

20. A method of treating a patient suffering from a 0*04 condition mediated by activated. T-cells, e-qg CD4' T-celln,. the method comprising administering Lo the patient an effective amount of a specific binding agent according to any one of claims 5-14.

21. The method of claim 20 wherein the specific binding agent is administered as a conjugate with a cytotoxic agent, in a cytoxically effective amount.

22. The method of claim 21 wherein the specific binding agent comprises a Fab, -F (ab1) or Fv fragment of a monoczlonal ncibody capable of recogaisirig O-0 agen when expressed on the surface of CD4- T-cells'.

23. The method of claim 20:wherein the- specific binding agent is an.immunoglobuliln capabl.e of cytotoxic effect, on cells bearing Ox-40 on their sturface.

24. The method of claimr 20 wherein the specific binding *agent can fix complement or mediate antibody-dependent cellular cytotoxicity. The Method of claim 20 wherein the specific binding agent has or is linked to structure characteristic of the Fc region of an imiunoglobulin of murine type IgG2a or human type Ig01.

26. The method of claim 20 wherein the patient-is suf fering from a condition -selected from the group consioting of-: multiple'sclezosis, earcoidosis, rheumatoid arthritis, autoimmune uveitiSG, T-cell lymphoma, rejection of A traiisplanted organ or tissue, graft -versus -host disease orreaction and inflamnmatory bowel disorder. 21. The method of claim 26 wherein the patient has graft-versu6-host disease or reaction. 28, The method of claim 26 wherein the patient bae an iinflamrnatory bowel disorder.

29. The method of claim 26 wherein the patient is suffering from multiple sclerosis.~ The method of claim 26 wherein the patient is suffering from earcoidos is. 044

31. The method of Claim 26 wherein the patient in Ip suffering from rejection of a transplanted organ or-- .00:0. tissue.

32. The method of claim.26 wherein the patient i s suffering from rheumatoia arthritis. *33. The method of claim 26 wherein the patient is suffexing from autoimrnune uets

34.. The method of Claim. 26 wherein-the -patient in suffering from a CD4* T-.celi lymphoma., A method for reducing a population; of T-cells, e.g. CD4' T-cells,. that express an OX-40 antigen in a human host, comprising administering an effective amount of a specific binding agent-according to any one of to 14. 36, The method of claim 35 wherein the specific binding agent comprises an antibd-toon cojugane which binds substantially only the Ox-40 antigen, administered in cytotoxically eff:ective amount.

37. A method of inhibiting relapsing autoimmunie inflammation in a patient suffering from multiple sclerosis, comprising the steps of administering to the patient a therapeutically effective amount of a specific binding agent according to claimas 5 to 14.

38. The method of claim, 37 wherein the specific binding agent comprises an effective amount of inununotoxin binding substantially only to the Ox-40 cell surface- Sol* antigen-

39- A-specific binding agent according to any one of claims 5 to 15 for use, in the treatment of a condition- mediated by activated T7cells, e. g .C04* T-cells.

40. A specific binding agent according to any one of claims 5 to 15. for use in' the .manufacture of a medicament for use in the treatment of condition mediated by activated T-cells e.g CDC~ T-cells-

41. A- polypep tide according to claim 3 or claim 4 fo1r use in the preparatilon of a specific binding-agent according to jany one of claims 5 to 0 too 0 *a,

42- A method ot preparing a polypeptide according to claim 3 or claim 4 or a specific: binding agent according to any one of clainS 5 to 15 which method comprisea culturing a host organism according to claim 18. 43 Use of polypeptides according to claim 3 and/or specific binding agents according to claim 5 in specific binding reactions in subjects of treatment with polypeptides according to claim 3 and/or specific binding -agents complemrentary, thereto. DATED this 4th day of November, 1998 Andrew Dale Weinberg AND Arthur Alan Vandenbark By DAVIES COLLISON CAVE Patent Attorneys for the Ap'1icants