CA2184780A1 - Methods of diagnosing and treating cancer - Google Patents

Abstract

The present invention relates generally to methods of diagnosing and treating cancer and more particularly colon cancer. Even more particularly, the present invention provides a genetically manipulated live animal model useful for developing diagnostic and treatment protocols for colon cancer. The present invention further provides agents useful for diagnosing and treating colon cancer in animals such as mammals including humans. In a particularly preferred embodiment the present invention provides a genetically altered animal or progeny of said animal wherein said animal comprises a mutation in at least one allele of Cdx2 or equivalent thereof and which is a homologue of the Drosophila caudal gene and wherein said genetically altered animal has a predisposition to develop growth of neoplastic cells in intestinal epithelium.

Description

son Ca~e 61 3 g254 2770 p.0d, r u ~ v ~ ~ 1 ~ 4 7 8 0 I

~IE~ODS OF DIA~GNOSING AND
~}~EATI:NG C~CER

5 l~e present i~vention le~a~es ge~erally ~ ~ethods of ~ ~d t~eat~n$ ~n~r ~nd more particu~arly colon ca~er. Evell more p~rticula~ly, the present i~velltion proYides a ge~etically manlp~lakd live al~imal model useful ~or ~.,elopi~ s~.ic a~d lre~ .cn~
protocols for colon ~noer, The present illY~ tiO~I further provides agents useful for nosj~ and treat~ng col~ cancer in animals such as m~mtnql~ including ~umans.
Bi~liogr~phic details of t~e publieatio~s ~lu~,licd~ly referred to in tbis ipe~if.ePtio~l ~e collccted at t~e end of the dcsv~ . Sequence Idc~tity Numbe~s (SEQ ID NOs.) for ~he slucleo~de and ~o acid s~J~ce~ ref~rred to ~ll ~e ~pee~ on are defined follow~ng ~e bibliogr~phy.
Tl~ougL~ b~ io~ and ~e Clalm6 w~ich f~llow, u~less tlle context re~u~res otl~,e. wiDe, tJ3e word "comprise", or ~ ;ous such as "co pli~es" or "~r..~;siug", will be ~ A~d to imply ~e ~lu~ of a stated integer or group of ~tegers but not ~e exclusion of ~y o~er i~teger or ~roup of intege~.
l~e i~ Jyh;~lu~ " of recombinant DNA ~ cb~~l~gy is g~eatly f~ esea~h ~d d~lo~ ent in ~ medical a~d ~llicd health fields. A particula~ly i~pOl~l field is ~esearch i~to the d~ of ~cers, such as colo~ e~. An ul~ls~ n~ of ~e rroler r and genetic m~çh~nicm$ lcading to the ~ J2~ of colon ca s is r~u 25 to f~ h~'A rational de~ign Of ~iiae~t~ rll p~t~ols.

C~I~ ~ and more sp~ifically colorectal c~ci-~ua develops ~om gcn~tirqlIy di~ecte~
stepw~e plle~lyp;~ alt~atio~ of be rpith~ m ~1). l'he g~D~t~ bas~ d the p~e~otypic a;l~Adti~ e diverse a~d affec~ tum~ UIIP~SO~ genes, V11C~eLI~S and mism~tch repair lW r~ ies Col l is~l Cave 61 3 9254 277~3 P.~î
~1~4780 v . 4~

genes ~nd leads to ~vasive carcinoma.

The h~)".oo~ ~s a coLs~.~ed DNA sc.~u~,lce encoding a se~uen~specific D~ bindinghomeodomain ha~ a~criptio~ factor pru~ s (2). C~ i.s one of tllree murine homologues of ~e Drosophi~a ho~ gene, caudal (3). The others are C~l (4) andt~-4 (5). In l)r~so~hil~, d~L.lll,~ of ca~dal ~r~ssioJl causes se~ere di~L ~ in body seg~e~ ioll ~nd posk;llor sl,ue s ~6). In the mouso, Cdr2 is e~ ~d extra-e~l)~ ~ieally at 3.5 day post coinlm (d.p.c.) in the t~v~he~ d lat~r in some trophect~ ally derived placental tissues. El~blyon~c e,~p~i<,n of ~2 be~ins at 8.S
10 d.p.c. ia the pOSl~iul ~Ut, the ~lbud a~d ~e posterior part of ~he n~ tube (7).

In wo~l~ le~ up to ~e p~ el~liO.~, the i~vcrltuL, sought to i~ e functtQr~l role of ~2 dur~g mouse eml~l~o6cncsi~ using genetically alte~ed mice. In ~he course of ~is in~esti~ti~n it ~vas su}prisingly obse~ved t~at mice h~ o~ygo-~s for (~dx2 developed 15 at high frt;~ Cy il~sl;l~t adc~,orl,~t~ s polyps. The ge~Lic~ly ~tered miceprovides a rnodel fo~ colon carc~noma în animals s~ch a~ m~mm~l5 hleludi~le huma~s and provides a ~is for ~ d,~ t vf a range uf~J;~n~ ~uLi~ agents for col~n carci~o~.

i~ly, ~- a~t of the present i~ t.iV~ J lc~ h S a ge~etically altered anirn~l or 20 progeny of sald anin~ av~ ~ ~sitio~ to tevelop ~rowth of ncopld~lie cells in ~ r~ l epith~ m More particular~y, the prese~ iu~ iO~ ~~t~ lates a ~n~ti~ y altere~ a~mal or pf~ y of said ar~ ri~ a ..~ ;on ~n at least one alle~e of a h~uolog~e of the Drosopk~
25 ca~al ge~e.

Even more particulally, ~e present invention i~ dirccted to a ge~ r~lly alt~red a~imal ~r prog~i~y of said an~rnal ~p~i~ a ~ ion in at leas~ one allele of ~x2 or equivalent ~eof and whi~ ~, a ~ ue of the DlVSGfJk l~r Ca~a1 ge~e and wherei~ ld gen~tirPIly 30 altered a~imal has a predi~,positio~ to develop growtb of neoplastic cells i~ ~ntesti~al 03-SEP-lg~16 1~:el3 DalJie~ Cc!lisorl Cd~Je 61 3 gz~c~ 27~a P.06 epith~lium.

The animal may ~e a l~bOf~lO-y ~est a~imal such as a mouse, rat, rabbit or ~guinea pig; a u,~ animal suc~ as a pi~, sheep, cow, ~orse, donkey or goat; a co~ n;~ nirnal suçh S as a eat ~ do~ ~r ~ oapti~/e wild animal such as ~ ~an~roo, deer or fox. Preferably. the animal is a labor~or~ test animal and is m~,t pl~Çe,.lbly a mouse.

~ "pre~ ion" to devclop ~rowth of ~opl~ ells ;DCIUdeS at least 1 in 100, more p)r~f~r~ly at least I in 50, even mo~e pref~l~bly at least I in 1~ and still even more 10 preferably betwee~ to g in 10 animals develop ~ ic cell grou~b ~ver from ~out 3 mo~tbs to about 2 years.

The g~n~t;~ y a~tcrcd animal contai~s ~ disl ~lptiOI~ to the expres~i~n c~r expression product of a ~roso~ki~ hon~ e of caudal. ~efe~ly, the hon~ologue is G~2 altbwgh ¢he 15 ~rese~t invention extends to equi~alen~ of th~s gene or h~ -lo~,ues of ~is gene. Most p~ d~ly, ~e gene LS murinc ~;2. ~e m~tant C~2 gene gcncrally ~ntair~ a single ormultiple nuclcotide s~Jl,~ iQ~-r addition and/or deletion such dlat the ~ene ts ~o longer transcribed or ~s lLL~ ed into a non-fuil length L~nsc,i~t or is i~p~ble of direc¢illg a wrmal ~u~ Al Cdx2 protein or is incapable of direct~ng sufficiPn~ production of normal 20 C~x2 protsi~. In one emb~il~r ~ e C~r2 alleles are deleted or su~t~ y de1eted by ~omologous lce~ oweve~? any of a ra~ge of ge~t~c al~la~ns may be induce~
~ ~;l;.~r rende~ the ~use or other g~ ly al?~red ani~al h~ ?vu~ ~2 mutants or heh,l~4rg~ur? Gtx2 mll~ntS.

25 The g~h C~f ~!q~ti~ cells is ~llerally in the form of i~testir~l a~N~ll,atous polyps and h~s is eo~ ul a form of, or a prelim~? form of? colo~ ciDama. Tumour growth in a h~fo~ C~x2 mousc may be chi~rac~ d by at Ieast one of the r.~llo.. ing:
~i) si~leo~ Ui~ f adenOD~L~U~I polyps mai~ly In ~r~ al large ~t~ r-; ~nd~or (ii~ large bowel masses ha~ing ~e a~peaxa~ce of ~ viUous ~1~ at the 3~ micr~ic level? andlor E~3-5EP~ 6 16:B0 DalJie~ Colll~4n C~e 61 3 9254 27713 P.0~
218~780 -~iii) crypt archi~ture grossly ~ LU~lJCd with ~bn~ 711y situated mitoses in dysplastic epithelialcells; alltlor absencc of Cdx~ e~?ression in epi~elial cells of colonic tumour6.

5 The ge~t;oally al~red aninaals of the present ~c.l~ion provide an ani~ mo~el for ca~eil.~...a of the colon or ~t least a ~r~u~sor stage thereof. More p~ l~ly, dle animal model is for human c~ ~nla of the colon.

Ac~,~li~l~, ano~er aspoct of ~e p~esent i~"~nLicrll pravides an anirnal model for carcinoma 10 of the colon, or a p~ee.u~or stage thor~of, said animal model comprisulg an animal or its progeny having a ~redisposition to develop ~ of neopl~s,tic cells in iJ~stîn~ pithelium.

More particularly, the prese~t iL~-~e~ ~pl~tes an animal model for carcinom~ of the oolon or a ~,~ur~ s~,e ~ereof, said a~i~l model co~ is~og a geJ~t;~lly alteret an~mal 15 o~ its prog~,~ comp~s~g a ~ in at l~ast o~e allele or homologue of the Drosophfln cau~t gene~

Eve~ more particulally, t~e prcsent iM~ention is di~c~ted to a~ a~imal model for Cal-Cil20ll~a of the colon or a ~ s~,e thereof, s~id animal model comprising a~ a~imal or its 20 progeny ~ mg a m~ on ~11 at least onc allele of C~c~ or e~uivalent the~eof and which ~s glP of the ~rosop~lila cauda~ gene and wherein said snimal has a predispositAon to d~velop growth of ~eoplastic cells in ;u~ epith~ ]m.

Most preferably, the present i~ n~ion p~o~ides a~ a~Amal model for c~Arcinoma of the colon 25 o~ a p~ ;, stage t~creof, said a~imal model c0~li4il4~ a mouse or its p~g~ having a ml-r~tioJl in at least onAe allele of dle C~ gene or i~ equivaletlt arAd which is a h~mclcg~le of a Drosopkila caudal gcne.

With ~espe~t ~ ~e lat~ p-~e~~l~t el~lbo~iAAAe~l of the present ~[~ ion7 the g~n~ lly 30 alte~od mousc is gener~lly a ~Z ~b~ mutant. IIV~ A~ apter tumour de~elopme~lt, EP--1~96 16:01 D~l~c Cclll5~rl C~,~ 61 3 ~Z54 27'7~ P.~3 ~e n~l~etir cells may be homo~ygous Cc~2 mutant, referred to herein as a null m~lt~tion or -J- mioe. H2tL~,4~gous mice are referred to as +~- miec. Normal mice are referred to as ~+ mioe. In accv.~nce wi~ the present inveIItion, -1- may be rescued which may provide an alternative or ~mpl~ ,t~r~ mouse model for col~ ce~.

Tt,e ge~etically altered an~mals of ~e present i~vention per~nit the dev~lopm~nt of a rallge of di~ ostic tests for c~ei~ a of ~c colo~l or a pre~w~sor ~e~of or a predispositi~n to devchp c~i,~...a of the eololl. Th0 p~esent i~l~en~ion is pred~ rd in part oAl ~he ~igh raoe of tumour devel()pmP.nt in Cc~ mice. Accordin$1y, SCA-U~ ni-ng for il~divAduaAs such as 10 huma~ for a ~ J~ in a Cd~ allele wlll ~ dde an in~ic~ n o~ t~e likeli~ood of cancer de~elopment AA~1 the colon. FOA- example, whcre ~ subject such as a hum~ subject has relati~es wi~ a hiseory of colon can~er, ~he subje~ sted G~A be SCl~A~AÇd fOA a ~ t5t;ol~
i~ an allele of C~. This wil1 provide a~A in~ n of whether f~milial eanoer is likely tc develop.
~CCtil~i~ tO this eAA~ there AS provided a meth~d of determining a subject s pred;~osilioll ~o ~:lv~viu~i~5 familisl C;il.~.iAu~ a of the &olon, said TAotAAod ÇoA~ isA~
A~ g a bh'c~ sample firom said subjec~ c4.~ nil~ oells a~ screening for the plv~llce of a mutatioll in a homologue of ~e OrosophA'la ca~dal gene wherein ~e presence of a 20 i~u~ ~i.... in at least one ~llole AS i~ldicative t~lt ~e su~ject is likely to develop c&.;ino,l,a of the colon.

P~er~Iy, the gene t~ be sc~ is Cd1~2 or i~ equivalent.

25 Sub3ec~s hav~ng mutant ~2 all~les are moro likely to develop ~olon ca~er.

The d.,~ All or lil~ç~ ood of d~,~vlop~-\c,~L of s~A~adic c~eiL~a of the CO10An may be oy obt~inin~ a biopsy from the i.~ .e a~d screen~ng for ~e pl~sence of a r,. J~ion in ~e G~2 gene. Again, ~e plc~ncc of a ~u~ in at least o~e allele LS in~icative of a ~0 strong ~ ihood ~at a nlmour w;ll develop in the col~.

03-SEP-lg96 16~ D~vie~ Collison Cave 61 3 9254 27~0 P.04 ~VA_ . ~v~ 218~780 .

~ccord~l~g tO this embodimellt, t~cre is provid~d a mcthod ~f diagnosing colon ~ncer or ~
likelih~d of developing colon cancer in a subject, such as a human subject~ said met~od comprising obL~i"ing a sample such as a biopsy sample or a sample ob~ained foll~wing surgery of ~e subjut's intestine ~d sc~ or the ~ ~ of a mu~tion in an allele of S a }~ ~lP of a l~ sopki~ ene such as a ~2 wberein the pr~se~ce of ~ m~ation in at le~st onc allele ~s indicative that colon cancer has or will develop.

The screenin~ may be accomplishod in any rlumber of ways such as using oli~o~ ot~
p~obes or prlmer~" polr~l~se chain reaction (~C~) analys~c s~ch as RT-PC~, dif~l n~ial 10 ~yl~- ;J;s~;o,- or ~u~L~,d protei~ a~alys~s.

, an im~u~logi~l test may be cor,d-lc~d of tntPstin~ tissue. According}y, the present invention extcnds t~ a~ i~ies to ~1 or part of Cd~2 for use ~ scl~, for the pr~senc~ or abse~ce of Cdx2 ~y~ io~. The anti~odies may be monoclonal or polyclonat 15 a~d may be f~"e,~t~ of al (e.~. Pab fragJnPnt.c~. The ~ntibodies nay also be ~,yntllclic or re~lnl7il~n~ or the a~lLil~d;c~, may be ~ybrid ~mihodiP~

The antibodies may ~ rec~d to r~bin~ Cdx2 or to a del;~t.t;~ thereof. A deriva~ivc includw a ~ l form tbereof, a r~ part thereof OI' a hlsion r lol~lP C0LUP~ &
20 all or part of Cdx2 and ~nother molecllle such as maltose binding protein or glu~iolle S-tl~r~

Thc G~ d;~s may be hbellet with a repo~er ~QI~x]ie capable of plvvi~iag U~ idcntifi~le sigrlal. Antibody binding is then d~t~d by seree~ fiX the reporter n~ llP
25 Al~.~ti~ binding is detected by binding of a second antibody di~ectod to s~id first a~ti~ody and labelled with a re~rter molecule Ac~ giy~ the present invention is directed to both r--~ ibodies.

Bo~ polyclonal alld .,l~l~clolla' antibodies a~c ou~inal~le by imm~ tio~ ~ith Cdx2 or its 30 d~ivatives and ei~ ~e is utilizable for im.. ~ s. T~e metho~s of obta~ning bo~ types 03-SEP-i9~6 16:~32 ~ies Cclli on ~a.)e 61 3 ~254 2770 P.05 218~780 P~71n\ElS~: X h rP~ n/ss . _ of s~ra arc well haown in the ~t Polyclonal sera are relatively easily p~epare~ by hlj~L;on of a suitable laboratory animal w~h ~ e~e~L~ amount of Cd~ or anti~e~ic pa~s thereo~, collecting ser~ from the animal, ~d is~l~tin~ specific sera hy any of the ~n~wn imn~t~no~sorbent techniques. Althou~h ~tibodies ylud~ced by this metllod are utili~able in 5 virb~ y any type of i~mu~ c5~y~ they are generally less favoured because of the potenti~l h~ g~e.~ vf the product.

The use of monoclonal a~tibodies ~ ~ ~u~oasi,a~ ig pr~er.Gd beca~se of ~e ability to produce them ~ Isrge qu~lilies and the l~om~enei~ of the product. The prepal~tioll of 10 hyb~idoma ccll lines fo~ mon~elc -t ~.Lil,~d~ uduclio~l derived by fi~sing a~ u~o~ c~ll line and l~ pho~,rl~ s~ ed ag~t the illlUIllJlO,~C p~eparation can be done by ~b~L~ ~ which a~e well IsIIown to those ~bo ~e ski~led in the alt.

~ other aspect of ~e present inve~o~ c4rt~ ~,pl~t~ a me~od for detec~ng Cdx2 in a 15 b ~ J~ sample from a sub3ect said method c~ e4~ g said bioloE~ical s~mple witb an antibody ~pecific for Cd~2 or i~ de.;,~ or homolog~3es for a time ai~d u~der conditions sufficiPn~ for a~ od~-Cdx2 complex to form, and ~hell det~ said complex.

The pr~l~e of Cdx2 may be ~ceQ~pl;~l-ed in a ~mber of ways such as by Weste~n blo~ g 20 and ELISA proce~es. ~wide ra~ge of ;.. ,~ y te~iques ~re available as c~ be seen by l~..~ to ~'S Pate~ltNos. 4016,043, 4, 424,27g and 4,01~,653. These assays also include direct bindin~ of a labe31ed ~tibody to a target.

1~ accorda~oe with ~e pxcs~nt inventis~ e sample i8 one which mi~ht contai~ Cdx2 such as 25 ctlls or cell e~ac~ and tissue biopsies. The choice of 4amplc will also depend o~ whether d~ of spo~a~ic or familial CanG~ is bei~g sough~ Spo~adic cancer would reql~ire a~alysis of biopsy sarnples whereas familial cance~ may be sc~ od u~ any cells.

By ".~1 1l;.A mr~~ cl11e" a~ used in ttle pre~ent specifiGation~ is mea~lt a lok~i! ~ whicl~ by its 30 c~.c~ic~l na~re, provides a~ analytically idt ~;fiA~le sig~al which allo~s ~e d~,'~tio~ of 16~ 5 ra~ les Co ! l ~son Cav~ 61 :S ~a~ 2~7~ Fn ~7 218dt780 r ~ ~tC'AN5.PI~.V 41~S

antigen-bou~d alltibody l~eteCtiQ~ may ~e either qualita~ve or qu~r,til~iYe. The most commonly used reporter mn~ is ~pe of assay arc cithcr e~y~ fluoiuphore~ or~a~io~uclideoo~ 3molecules(i.e radioisotope~ d~h~m~ min~c~ntmolecules.
I~ the case of an enzyme ~mm~ ayl an e~me 3S co~jugated to ~e so~ont an~i~ody, 5 ge~erally by mearls of glutaraldehyde o~ periodate. As will be readily recogniLed, however, a wide varie~y of diff~ent c~ju~;alion ~chniques exist, ~hich are readily a~ !able to ~he skilled altisan. Cont ~ ly used ~ c~ i~clude hor~e"ldish pero~idase, glu~ose oxidase, ~-~al~ctosidase a~d alkaline ~hnsph~tacçr amongst others. ~be subs~es to be used wtth the specific eazymes 0~e ge~ ally chosen for dle pl~od.,~ , upon hydrolysis by ~e corre~ponding 10 e~zy~e, of a d~ ,~ble colour chan~e. Exunples of suitable en~ymes include alkali~e ,k ~c a~d p~dv,~;~d3e. It is also possible to employ fluoro ~enic subs~a~es, which yield a fl~o~ ,e.~ oduct ra~er tha~ t~e chromoge~ic subs~ates noted ~o~e. Itl one metho~, the enzyme-labclled a~ti~ody is added to the ~Irst sn~bod~-Cdx2 ~ I~, allowed to ~d~ and then t~e e~cess reage~t i~ washed ~way. A solu~on C4~ 3 the appropriE~e DI~ e is ~en 15 added eo ~e o~mplex of a~ody~ tige~ L~odr. The sul~ will react with the e~ym~linked t~ the second ~L~od~, giv~ a qualitativc visual sig~al, whieh may 10e fu~tber q~ y ~e~ ,L~ ~ome~icallyt t~ Ftive an in~ ;on of ~e a~nou~t o~ hap~ which was p~esent in the ~ample. r~e;porter moleçule" also extends to usc of cell a~llltit~tinrl or inhibition of R~&ll~t;l~ioll such as red bl~d Gells on late~ beads, ~nd the lil~e.
Al~e.~it;ly, nu~resce~ com~ounds, such ~s fluorescein and Illod~"h.cl snay be c~emically c~upled to aL~ i; without alte~ing theu bi~ding capa~ity. Urh0n ~ti~a~e~ by il~l~min ~ion wltb light of a particula~ wavelengtll, the fluo~chi~Luc-labelled antibody adsorbs the light energy, hld~ , a state to e~cita~ility i~ the molecule, foliowed ~y en~i~sion of ~e li~t at a ~5 characte~iseic colou~ vi~ually detec~ble witb a li~ mie-~soope. As in a~ e~yme ir~muuoassay, the fluo~ t la~elled a~tibody is allo~ed to bl~d to the first antibody-Cdx2 complex. Af-er ..~hi~g of~the unhountl reagent, the r~ A~ tertia~3r o~ rl~,A is ~e~
e,~oscd to tbe lig~t of the ~pplvp~iate u~avcleng~h the fluorescence ohgG~v~d in~i~?tAC the presence of tbe hapte~ of inte~est. Immu~on~.on. y,r~nç and e~zyme in~uno~say Pchniques 30 a~e bo~ y well estab1ished ~ the art and axe palti~arly pr~f~ rr~d fur the p~esent metllo~.

EP-lg~6 16:04 l~ '~ollison Ca:~e 61 ~ ~1254 2771a P.el8 NC?1tV.~9~

~.

vG~, other reportcr molecules, s~ch as radioisotope, chemilll~inesce~t o~ biolu...inosce~
molecules, may also be e~ployed.

The present i1l~entio~ also co~l~."plaees ge~etic assays wch as in~olving, for ex~ n~le7 PCR
5 analysis to detect C~2 alleles o~ t~eir ter-~ati~t~. A~ dti~r~ methods or ~otl~ds used in COL~J~ a~ysis i~clude direct nucleoeide sel~u~nri~g OF m~ltr~ )n S~~ ~nit~g SUC12 as singlo seranted confo~m2tion poly~o",~ous ~alysis (SSCP), spccific o]i~G~ c~olide hybx~ic~q-ti~ or ~ciho~l~ sllC~ as direct protein ~ e~tl0J- tests.

10 The prese~t ~nventiorl is fur~er d~ted to an isolated nucleic acid moluule comprising a ~qmm~iq.~ homologue of dle Drosophila c~a~ ~en~.

A mammai as contemplated above is as here~before dc~lned but does not extend to a mouse h~ olc~ when the homologue is C~2. ~is aspect of t~e present iJ~ lliOI~ is desc~ibed S h&G;.a~ r with l~fer~ to the huma~ hnm~og~ of Cd~c2. This is dvne, however, with the ,.s~ling that ~ vl~u~les in other ~on-murine animals are con~ d by the present illV~ O~.

Acc~ gly, a~ot~er aspe~t of the prese~t i~rtio~ con~l~s an isolated nucleic acid20 mol~ule correspo~ding ~ a hum2~ hom~ogue of ~e Drosoph~la caz~ gene, Cdx2.

T~e nudeic acit mo~ ~' of ~is aspe~t of ~e prc~ent invention e~codes Cd~2 and c~nprieeS
a sequence of nuc~eotidci, havi~g at least about 60% ~imil~n~ to ~e nucleotide sequellce set for~ SEQ 1~ N0 :1 and which is capable o~ hybridisi~g under low stringency conditio~s, 25 ~t 42~C to the nuclea~de sequ~nce set for~ irl SEQ ID N0; 1 The prese~t h~e~liO~ also p~ovides a nucleic acid ~olocule which encodes Cd~2 and co~l~ ises ~ se~ n.i a- of nucle~ti~es hav~g at least about ~0~fO similari~ to the nucle~tide se~ Pn~e set forth in SP,Q ~I) N0~ d which is capable of ~ ti~l;sillg u~der low- st~ hcy ~oud;l ;~n~, ~0 at 42-C ~ ~e mlcleotide ~ F~n~ S~. for~ SEQ ~ N0.~. The 5~ e 3çt forth in SEQ

03-SEP-19~04 rla~ i es Co 1 1 i son ~a~ e ~;1 3 ~5~ 277~ P . 0~1 r- I ~C.PIV.~
_ _ - iO-NO:~ is a ~e~omic 5~1~ence for murine ~2.

~he nucleotide molecule i9 preferably d~ ~lc from the buman genome but genomes a~d nvcJe~lP &~ ~5 fi~m no~-murine a~imals are also e noompqs~ed by the present invention.

Refe~ence herein to a low ~hi~g~ at 42~C includes and e~c~p~s~s f~om at least ~bout 1%
vJv to at least about 15% VtY fonn~ ni~ d from at Icast about IM to at least about 2M salt fo~ hybri~ic~ n~ and at le~t about IM to at le~ss about ~M salt for w~eh~lg cQnt~isi Al~l.aliv~ Sain~n~ ;o)~c may be applied where nec~ ! sueh ~-c medium strin~ency, 10 which i~cludes and ~con.F~s~s f~om at le~Let about 16% v/v to at least about ~0% vlv f~rmamide and ~rom at least a~out 0.5M s~ at least about 0.9M salt for ~ybridi~h~n~ and at le~t about O.ShI to at least about 0.~ salt for w~Li,l~, conditions, or high s~ ency, wbich i~cludes and ~ om a~ least abou~ 31% vh~ bo at le~t about 50% v/v formamide a~Ld f~om at least about O.OlM to at least ~boue 0.15M salt for h~b~ all~n~ and at lea~t about 15 O.OlM to at least about 0.1 S~I salt for was~g con~ iolls.

Accotd~ly, another aspect of the prese~t inv~."io~ provides a melhod for c~on~g a nucleic acid mnl~-u'e P~co~u~ human ~à~2 or a ~ ivd~ th~eof ~r ~ par~ theroo~, said method comprisi~g ser~g a nucleic acid library with mouse Ccb~2 I~N~ or ml or an 20 ol~nl~rl~otide probe ba~ed on r~ousc Cct~r2 D~A or mRNA. ~e me~hod of ~is aspect of the ~resent i~ tio~ may also use PC~ to ~lone t~e tar8et nucleotide se~ ce.

further aspect of the prese~t illve ltiOII eQnt~nplates a method for elo~ a nuclootide s~ ei~r~ g a Inu~an Ca5~2, ~aid medlod cc~,pris~; searchi~g Q nucleotide data base ~or 2s a sç~l~ce which e~codes a molecule ~ least 60% ammo acid ~jmil~ity to mouse Cc~r2 or u~ere the oucl~ti~ s~ is at le0st 60% ~ ilar to the mouse Gcbc2 gene~ de-;gniug one or more o1~ ~Iide pnme~s based on a ~ eo~e s~l~.c.~r~ loc$ed in the sea~ch, sc~
a nudeic acid libra~ ~rith said o~e or more o~ çleotit~ d obtai~ng a clo~e Lh~l-,rlu~
which e~es said human Cd~2 or part tbercof.

0~-SEP-1996 16:05 ~a~le~ Ccllison Ca~e 61 3 9254 277~ P.l~
218~1780 pl~ ~ r~lRV 4~96 Prefe~ly, the nucleic acit libra~y is a cDNA, genomie or m~N~ libra~y.

P~.~ly, t~e ~IJcleic ~cid library is a c~NA e,~r.,3iion li70rary.

5 Ano~er aspec~ of the ~veD~ion is ~irected to the human ~Rr~ic C~2 Bene a~d to 3' and ~' regions ~rcof a~ well ~s fi sion molecules beh~een C~i aIld other molecules su~h as mal~ose bindin~ protein a~d ~ut~thione-S-~srerase.

S~ll sno~ Pmbo~ n~n wntemplates the pron~o~el or a filn~tir~n~l part thereof of ~he human 10 genomic Cc~c2 13ene The ~ t~ may ~eadily be ~,d by, for example, "ch..,r"oso."e wall~i~,".

Another aspec~ of the prese~t ~ e.,~ion is directed to ~uman Cd~ prote~n ~cluding a ro~oi~...~ form tJ~ereof h~v~ a~ o acid ~ e of at le~st 60% similarity to mouse 1~ Cdx2, set forth in SI~C~ ID NO:~.

Otherper~ ge similari~es at the nucleotide or amino a~id levels i~clude about 70~~'o, about 80%, about ~0% and about 95%.

20 ~e pres~t i~ crnhon fi~b.e~ Gon~rl ~tt~s a range of der~va~es of human Cdx2 protei~ and its gelle~c sc~uent es D~.iv~tv~ i~clude r.~ c, p~s, portions, mutas~ts, homolo~ues and analo~es of ~e human C~x2 polypeptide and co~ ondin~ ~enetlc sequer ee. I:)eriva~ives also indude si~e or multiple amino acid gu~ ;hll;on~5? deletions andlor adt~o~s to Cdx2 or s~le or mllltirle m~ itl~ s~ tonc, del~tionc andlor ~dditiv~ he Cc~c2 genetic 25 s~qut~n~ ''~d~ n~ 0 amino acid seq~ or 1nlcleoti~ se~l~eL~ces includ~ fi~sions u ith other pe~ides, pol~p~tdes or pro~s or fusions to ~uclcotide seque~ces. Reference here~n ~o "huma~ includes refe~ence to all d~liv~Liv~ thereof inr.~ ing îunc~ion~l de~ivati~es or Cdx~ immlln~ltJ~ y iu$~~Cli~ dc~

SEP-1~195 16:~5 Da~ies Colliso~ Cave 61 3 3~54 2~7~ P.11 TC~c.~ 6 2 1 8 g 7 8 0 ~nalogues of C:d~ contemplated herei~ indude~ but are not limited to, modifieation to side chains, incor~ th~g of unnshlral ami~o acids andlor their deriv~tives d-ring peptide, poly~ Jtidc or protein sy~thesis and ~e use of crosslinkers and otlle~ ethods ~ hieh impose conforn~tio~ ts on the proteinaceous molecule or their ?~
s Exsmples of side chaill modir,c~io~s oo .~..lpla~ed by the present invention include modificatit~ns of ~o groups such ss by redu~ive alkylation by ~eaction w~th an aldehyde follow~d by ~ Loll ~ith NaB~4; aa~idi~tion with ~u ,id~te, acylation with ace~ic ~hr~;dc, ~oyl~tivi~ of am~ groups with cya~ate; ~ u~cua~l~ion of amino ~roup~
10 wit~ 2, 4, ~trini~obc~ c sulpho~c acid (TNBS); acylation of amino groups with succinic ~nhydride ~d t~ ,pL~alic dllhy-hi~e, a~d pyridc~xylation of Iysine with pyrido~ 5-phosph~te foL~wet by reduction with Na~H4.

rhe guanidine group o~ ~e residues may be ~odified by ~e ~on~.~lion of het~ocy~,lic 15 c4nl:te~ p~ u~ whh reage~ts such as 2,3-l)ul one~ phc~ylgl)ru~al and glyoxal.

rbe ca~boxyl g~up may ~e modified by u~rbodiinlide activatiou viu O-acylisourea f~rmatio~
followed by subsequ~t dcr;vil;sation, for ~A~lll.le, to a collcsp~,-d~ amide.

20 S41~hyd/yl groups may be modified by ~O~c such a~ o~Luet~yldt;on wit~ i~dca~et;c acid or iodo~ e, ~.ru,~ c a~id o~dation to cysteic acid; fo~m~tin~ of a mixet ~I;sulrhi~Ps ~ith other thiol col~-~ou~ds, reaction with m~l~Pimi~, ~aleic a~hyd~ide or other suloeti11~tPd maleimide; folm~liol of merw~ial derivati~es using 4-chlorome~c~il~ , 4-chlor~ hpny~ p~ nic acid, pL~L~cA~u-~ chlo~ide, ~cblolul~.Wli-4-h1tloP~ o 25 and other m~r~als; ~o~moy~Sion with ~yanate at alkali~e pH.

T, ~"~pL~ residues may be modified by, fo~ e~pl~, oxida~ioll wi~ ~-bromo~lcrinimi~e or alkylaticn of ~he uldole ~ 2-hy~ .y-S~ b~rl broraide or sulphe~yl hatides.
l~rosiine residues o~ e o~r ~d, may be ~l~ed by ni~ati~n w~2 t~,~ih o!n~th~n~ to foxm 30 a 3-~hot~luslne d~i~c-Lv~.

03-~EP-1996 16:06 rlal~ies ~olli5c~n C~ve 61 3 ~254 ~7~ P.12 r.~ ~ANC~V ~J9,~ 218 4 ~7 81~

- 13~

Modification of ~hc imid~70le rin~ of a his~idine residue may be acGomplished by alicylation with iodoacetic acid d~, ;v~ ~ or N-c~ l,cll.vA.yla~ion wit~ c~ a~e.

~xamples of in~ ol~ting unnab~ral amino acids and d~i~llLiv~ during peptide sy~thesi5 5 include, but ~e rlot limited to, use of.o.~rl~ucine, 4-amino butyric ~cid, 4-amino-3-hydroxy-5-phen~ oic acid, ~-~inohPy~n~ic acid, t-butyl~lyci~e~ norvaline, phenylglyc~ne, or~ " sarcosine, 4-amino-3-hydroxy-6-me~hy;h~oic acid7 2-thie~yl ala~ne and/or D-isomers o~ t of u~l amino acid, ~omP~rl~ted herein Is shown in T~ble 1.
Clo~L~uA~ ca~ ~e us0d~ for e~cample, to stabili~c 3D co~ Qn9, using ~omo-bifi~ctional crossli~k0~ such as the bifu~ticnal imido esters hari~g ((:H2~ spacer gro~lps with n=1 to n=6, glutarhldehydc, N-hydrox~ucr~ itle esters a~d hetero-bifi~c~nal reagen~s which usually co~t~in a~ amino.reacti~re moiety such as N-hy~,,~lc~ io~ ide d~d anotl~er group 15 specific-reaetive ~oiety such as maleimido or ditbio moiety (SH~ or carbodivmide ~00~1).
1~ additio~, peptide~ can be ~rlnfio7m~tio~ y ~nstrained by, for e>~ ~r~>r~lLio~ of C,~
and N,-methylamino acid~ vd~elion of double bonds betwccn ~ atoms of alnino acids and t~e fol~"~ n ~ cyelic peptides or aIlalogues loy i~ du~.;"g co~ralent bo~ds such as for~i~ ar~ amid~ b~rul betwee~ the N a~d C tennini, betwee~ nvo side chains or between a 20 side cha~n and the N or C termir~

These types of modific~tioQs tnay be i~p~ to s~abilise Cdx2 i~ 3A~ini~t.ered to ac~.
individual or fo~ use as ~ di~gni~tic ~ ent.

~5 Other d~liv~ c~ ,~plated by the prese~t in~ention ~clude a ra~ge ~f glycosylation varian~s from a ~lpl~tcly ~l~cos~lated molecule to a modif~led ~Iycosylated molecule.
~Itered ~Iyws~ia~n patter~ may result from ~ ion of recom~ina~t molecules in different ho~t cdls.

03-SEP-1~6 1~:0~ D~lies C~llison C2~e ~1 3 3~54 c77~ P.13 v.~9.'915 2184780 ,~

The present ime~tio~ further co.~t~ plate~ ~mic~ Alogues o~ Cd~ Gapable of acting as a~lagoni~t~ or agoI~ists of Cd~c2 or which can ~ct as functio~lal a~alo~ues o~ Ct~. Chemi~
analogues may not necessalily be derive~ f~om Cdx2 ~ut may share ce~ain conform~tio~l simila~ ; Alte~natively, rh~,~ie~l ~n~k~gups may ~e spe~ircally de~tgn~d to mimic certain 5 physi~~ plvp~Ll~ofCdx2. Che~i~al 'o~;upsmaybech~n~ y~y~th~is~do~may be detected followin~, ~r example, natur31 pxoduct screenin~.

The id~ ,L~ ;on of ~dx2 permits the generatio~ o~a range of therapeutic ~olecules c~pable of mod~lr'i~ expressio~ of Cdk2 o~ modulati~l~ the ~tivity of Cdx2. Modulators lû con~e2llplated ~y t~e prcsent inve~tio~ include a~onists and ~t~o~ ts of ~ e~ression.
~nt~nistS of C~2 e~l~sion include ~n~ e moleGu~ ibo~ymes and c~ 3.~ ion moleGules. A~o~ts ~clude molecules wbich ~rease pro~ote~ ability or il~ ~e with negative re~latoxy mect~ mc Agol~lsts of Cc~ clude molecules which o~ercom~ any De~e xe~ulatory ",~,~ .. Ar ts~gnniQ2S of Cdx~ include antibodies s~d inhibitor peptide 1 5 ffagl~lerltS.

03-SEP-1~96 1~: 07 ~av ies CG 1 1 Isor- Cel~e ~l 3 9254 ~7713 P. 1~

TABLl~ 1 NC~A~.'ConVeA-Ational Code Non-con~entional Cc~de amino acid ~o acid t~-am~nobu~i~ acid Abu L'~-A~lethy~ me Nmala ~t-amin~-~-met~yl~outyl~te Mgab~ I~-N-r,~ yl~iA~ine Nmarg an~Aocyclopl~A ~ e- (: pro L~ methy~aSA.~dgA~AÇ ~m~sn Ca~ )XY1&Le L-N-methylaspaltic acid Nmasp 10 ~min~is~b~Lylic acid ~ib L,-N-methylcysteAne ~mcys n~l olboA~yl- No~b L-N ~ Aylglut~AiA~e Nmgln c~bo~Lylate L, N-methyl~lu~amic acid Nmglu cyclohexylalani~e Che~a L-~-methyl~;cti~line ~mhis cyclopentylalan~ne Cpen I~-N-methylisolleucine NnoAile IS D-alanine l:~al L-N methyllcuc~e Nmleu D-ar~e Dar~ L-N-meS~yllysi~e Nmlys D~spa~c aci~ ~asp L-N-me~yl~e~i~c Nmmet ~-cystei~e Dcys L-N-~yl~orleucine Nmnle D~glutrtline Dgl~ l--N-mett~yln~ e Nm~va 20 ~-ghlt~mic a~id l;~glu L-N methylo~ithine ~morn ~-histidi~e Dhis I,-N-s~e~yl~ lalanine Nmph~
D-isole~ine l:~ile ~-N-me~ylplo~ e ~mpr~
D-leucine Dleu I,-N-~ethylsenne ~mser D-iysine Dlys L-N-methylLl.rcollil,e Nmthr 25 D-~t~ r~h~c ~et L-N-mcLhyl~yt~ph~ ~mtrp l )-ollliLh;l-e Dorn L-N~ elL~lLyio~c Nmtyr D-phenylal~ine Dp~e L-N ;~}e~ylv~line Nmv~l D-p~oli~e ~pr~ L-N-methyle~ylglycine Nmetg D-~e~e ~)ser L-N-meehyl-t-butylgly~ule I~mtbug 30 D-~reon~ne Dthr t-~orle~ciIle Nle 03-SEP-1Y9~ 16:07 D~!ie~ Collison C~ve 61 3 g254 2~ P.15 r~ Rv ~
, D-tJ.yl~topl~ orvalitle Nva D-tyrosine Dtyr a-methyl-an.i.. o;sobutyrate ~b D-val~e Dval ~ ct~yl-y-am~o~ dte ~gabu D-a-met~ylalaD~e Dmala a me~byl-iycloh~ylalani~e Mchexa 5 D-~-met~yl~;~ç Dmarg a-me~ylcylcopenblala~c Mcpen ~)-a-m~ & ~agine ~masn cc-methyl-a-nap~ylalanine Ma~p D-cc me~ylasl,~ Dn~asp a-methyl~nicill~mine ~pen D ~ ~.ethylcysteisle Dmcys N~ n;l~bulyl)glyci~e N~lu D-~-lu~ly~gl~nq~ine Dmgln N-(2-&r"ino~ l)glycinç N~eg 10 D~ ylhis~d~rle I)m~is N-(3-~ op~opyl~glyc~ne Nom l)-~-methylisoleuci~e ~mile ~I-amino-a-me~yibu~r~te Nmaabu D-a-methylleucine ~mleu a-~ap~ylalanine ~ap l:)-a-methyllysi~e ~mlys N-benzyl~l~cine Nphe ~-a-methyL-Iell.;or~ e l~nmet N-(~ yle~yl)glyci~e Ngln 15 D-~m~h~lo~ l~c Dmor~ ~7-~ca~bamylme~hyl)glycine Nasn -me~rlph~ylal~e Omphe N-(2-~ yl~sly~ine ~Yglu D a-ale~ l~ne Dmpro ~-(c&rlJ~Ay~l~yl)glycine Nasp D-~-~e~ c ~mser N-c~clo~ yl~lycine Ncbut D-a-l~c~ r~ni~c I~m~r N~r~ollcplylglycine ~chep 20 ~-Q-m_~yl~yp~phan ~m~p N-cy.,lo~ yl~lycine Nche~
D-~-me~ yl~sine Dmty ~-cy-,lode ;ylglycine Ncdec D-~-methylv~ e Dmval N~ylcododecyl~lyc~e Ncdod D-N-me~hylalanine Dnmala N-cycloGclylglycine Ncoct D-N~ Ly' ~ iLe D~ar~ N-cy~lopruodglycille Ncpro 25 D-N-me~vl~s~zr4~ e ~mas~ N-cycloundecylglyc~e NcuIld I:?-N-methyls~l~ l)nma~ep N-(2,2-dlphcnyle~hyl)~lycine Nbhm D-N-methylçyste~ne Dnmcys 1~-(3,3-diphe~ l)glyc~ne N~he D-N-methyi~h1t~nine DNn~ 3-gu~ni~ h"r~l)glycine ~a~g l~-N~ yl~ . D~glu ~I-(l-hy~llo~y4~yl)glycine Nthr 30 ~ -methylhiet~ ne 1~, ~h~hu~rl)~glyc~ne Nser 1~3--SEP--1~96 1¢5:~ D~ule Collis~n C~ 61 ~5 ~25~ 2~ .16 r ~ Nc P~V ~ 2 1 8 4 7 8 0 - ~7-met~ylisoleucine ~)nmile N-(in~id~z~lylethyl))~lyci~e Nhis D-N-methylleucine l:~nmleu N-(3-indolyl~ y~ ycine M~
~-N-met~yllysine Dnr~lys N-met~yl-~-a~in~ e Nmgabu N-~2~yl~;yclohe~l~1aDine Nmche~a D-N-me~yL~io~ e l:)nmmet 5 D-N-mçthylo.. ;~e D~mom N-methylcyclopentylalanine N~cpen N-me~hyl~lyci~e ~Jala D-N-me~yl~ ylalani~e Dnmphe N-n~ y~ oisv~lr z ~ Nmaib ~-N-methylproli~e Dnmpro N-(l n.cthyl~l~,pyl~lycirle Nile ~-~-~ethylserine Dnmser ~-(2-l-~e~ ,~pyl)~lyci~e Nleu D-~-meL),l~l~o~ e ~nm~
N.,Icll~yllly~ ~ Dnmtrp ~-(I-met~yle~yl)glycfne Nval I~-N-me~hylt~lv,,~.le I~nmtyr N-methyla-~lalan~e I~ma~ap D-~-~ethyl~ali~e Dnmval N-~c~yl~ ;çill~mine Nmpen y-~i~ol,ulylic acid Gabu N-G~-hydro~yphenyl~glycine Nhtyr L-t-butylglyci~e Tbu~ N-(thiomethyl?glycine Ncys I S L-ethylglyciIIe Etg pen~ ~;ne Pen om~phenylala~e Hphe I,-~-methylalalu~e . Mala L-a-methylargi~ine Marg L-~-methyl~spara~ e Masn L-~ met~yI~ Ia~,p L-~-methyl-l-b~ lycine Mtb~g L-a~ yl~lrme Mcys L,-methylethyl~slyG~De ~etg 20 L-a-mcthyleluPrnine Myln L-~-me~yl~ll,t~ M~lu L-a-methylh~ e ~1i6 L~ me~ylh~op~ lala~e ~hphe L-o~methylic,~leuc~e ~ile ~-(2-methyl~ioethyl?glycine Nmet L-a~ ylleucine Mleu L-~me~yllysine Mlys L-~-m~yl~ onine h~net L-a~lne~ wP ~le 25 ~-a~nethyl~o~vali~e Mnva L-~ orn L-a-methylph~ e ~Iphe L-a~ ylpluIL~e Mpro ~a-~lly;~l"c I~Ise~ L-a me~ r~oniuc Mthr L-l~metL~lhf~ h l-~7 M~rp l,-a-l~c~ylly~ e Mtyr L-~-me~ylval~e ~val ~N-metbylhom~phenylalar~ine r~mhphe 33-SEP-lg96 16:0~ D~vies Collison Ca~Je 61 3 9254 2~713 P.17 F.'~,__, .' T~N.. PI~V ~,C/96 2 1 8 4 7 8 û

~-(N-(2,2-d;~ke~ l) Nnbhm h~-(N-~3,3~iphenylpropyl) ~nbhe car'oamylme~yl)41ycine ~a~bamylmethyl)~lycme 1-ca~boxy~ 2,2~henyl- Nmbc e~ylami~)cyGlo~,vp~e s Anotber embodiment of the prese~t .~Vc~ co~t~ s a method ~or modulati~g ~-~iu~ of G~ a huma~, said me~od co~r;~lng cor~ ting thc C~Z gene encotillg Cd~Z with an effective amount of a ~odulator of C~r2 c~p,~;on for a time and under 10 conclitions suf~lcient to up-~egulate or down-regulate or otherwise n~lodulate e7~pression o~
Cc~. For t~ &le, ~ ~ucleic acid molecule ~nr~ Cdx2 ~r a derivative tbereof may be in~odu~ ~n~ a ~ell. Co,lY~ " Cc~r2 ~ ee s~1~c~ suc~ as oligon~lcleotides may be irltroduced to ~e cell.

lS Ano~ ~spect of ~e present ~ OI~ oontemplates ~ metbod of n-fldl~Jrti~g activity of Cdx2 u~ a human~ said met~od cc~ ~g a~ t~ ;l.g to said ~ammal a mod~llq~irtg ~tive ~ount of a ~t~ e for a ~e and ~der c~l3dition~ s~ffi~i~7t to i~cxease or dccrcdse Cdx2 activity. T~e ~ole ~le may be a protei~aceous ~olecule or a ~h~ie~J entity and may also be a d~ iv~, of C~2 or iOE reup~r or a che~cal anaIogue or l~v~c~tin~ mutant ~ Cdx2.
20 Tho Cd~2 mnle~.le may ~uire o~ iori to pe~ bal~spo~l of the m~lecule ~rough the various ~e:~bra~ e cdl.

In a par~icularly pl~f .~ed embodiment, ~he Cc~c2 ~elle or a f;lnc~ e~coding nucleotide s~u~ ~Ge is in~oduced to a cell via a vaxiety of gerlotic mea~ h a~ by ~riral vec~r.
T~e iden~ficatio~ of the role of C~2 in ~olo~ cance~ d~,~p~ pe~i~ a r~ge of di~ros~c applicatio~s as hc~ befol~ desc~ibed ac well as oppu~ f~r ~ene therapy~

03-SEP-1390 16~ D~e~ Collison Oaue 61 3 ~1254 2~7~ P.1~3 Ig The prese~t in~ is fi~r ~e~ib~ by the followin~ non-li~iting f'tgures a~d examples.

In the fi~e~.

Figure I is a r~res~ o~ show~ ~e ~wle~:;on of C~c2 mutant mice.

(A) SchPn-q,tic r~o~s the er~tlo~nnus mouse Cdc2 locus (top), C~ eting vector (middle) and mutant allde (bot~om). C~2 c~ding exons a~e sh~un in black boxes PG~-ne~
is indicated by the stippled box. The 5' ~xte~al ~d intemal ~eo probes used fo~ ge~omic 10 Southern analysis of ES cell oolonies ~see belcu~ are s~owll in the ~lack li~es. Arrows indicaLe the p~ pl, p2 and p3~ used for g~ol~Lug pr~ge~y of ~t~;cy~vt~ mati~gs by ~e polymera~e chain rea~tion (PC~)- see Table 2 ~ lr;cl~io~ enzyme site~: B, ~ I, E, E:co~I, H, ~IrnallI, ~1~ NcoI, S SacI. T~e Xba~ res~ on site was ~troduced ~uring coll~lruction of ~c~2 ~,eL~ vect~r The ba~ ial neo a~d selection cassette dnYen by a 15 P~ h%Ir~ 5eP1L~ GK-no~), tog~erwit~ 4 kilo-base-pairtk~) 5' and 5.4 kb 3 ' f~ng s~ ~ serYed t~ replace 5.9 kb of the ~ative ge~e. Tbis invol~red the whole of t~e open reading frame (in~ ing the h~b~ mo~ , exc0pt for ~e f~l~st 136 ba~e-pa~r (bp3 G41B was used to identif~r ~, ,yc~ r~l clones.

20 The l~ g vec~was P~ t~ t el~ t~d in~ W 9.5 ES cell line. ~er l l day~
of G41~ s~;on 912 dmg ~ s~a~t ~ nie~ e picked a~d subsc~ ly analysed by Sout~em analysis. ~L~e clones yîeld~d the e~ ic~i~n r~ . Two were select~
~ produce ~him~c mice by ~n~cro ~jeclio~ inOo C5 7BL/~xC57BLf10ScS~ ~ost b1~loc~Male chimeric a~imals we~e mated wi~ C~ 6 ~ obtai~ m line ~AnQtnis~io~ of the 25 mutallt ailele.

e~ l~h;c~n frag~ent lengths iden~le~ in wild ~pe (WT~ d mutant ~T).

~ C) S~uthern analysi~ of five out Qf ~e C~ target0d lines. llhe left lane shows ~ wild-30 ~type W 9.S ~eU line a~ L~.u~ L-- show ~rgeted cell l~nes. T~ fi~e lef~ is ~findlll~ba 03-SEP-1996 16:0~ D~ies ~Dlli~or 0~ e ~1 3 9254 ~770 F.l9 F ~ r 2 1 ~ ~ 7 8 0 ~0 digested DN~ and ri~ht is ~o~ digested ~N~ hybrise~ with the S' e~te~l probe Bottom is the sa~e ~.,-l~r~, ~ybridised wi~ noo probo.

~ D) PCR assay uset to g~"ol~e 3.S d.p.c. ~I~Jge~ of het~z~ lc~loss~s show~ng S ~ree possible ge~ot~s ~ild type (~/~?, h~t~Gtc ~+~ d hon~o4Yg~

Blastocyst~ were ~ushed from the ~teruj and COnC~ US55 older thall S.S d.p.c. ufere t~l free ~f ma~Dal ~issues. To isol~te I; NA fo~ PCR, bo~h emb~onic and n~o~ -t~l tissues were Iysed Ul a ~on-iollic Iysis bu~fer such as PNDB Iysis buffer (~NDB ~uffer 10 uon~ins KC~, Ths-~CI~ 12.6EI~, gel~tin, Non~d~Q~ P40 and T~aen 20~ Witll prote~
K ~100 ~gJml) at S6~C. lPCR mi~ture w~ d ~ee pri~e~s sh~ Fi~2re lA;
pl ÇS'- TMAAGrCAA~ C~OMTCG -3' ~S~Q Il:~ N0:3]), p2 (S'- ~TA~I~TGA~GAGC~G~G~C -3' [~EQ ID NO:41)~
p3 (S'- GGGACl'~TCA~GTAC~OaA~ -3' lS~Q ID N0:~J~.
15 Rea~ion co~;lin"i~ were 96~C for 30 sec., ~S~C: for 1 m~nute a~d 7~DC f~ 3 mim~es for 35 cycIes i~ a 50 ~ on~a~ ~ 0.2 ~uM Of each p~imer ~d ~.2 mM of each of dNTP and I~CR 1ouffer (~iboo). A ~6 b~ ampIificatio~ prQduc~ ,ne~ted ~r~m tbe mu~t allele between ~e ~eo pr~mer p2 a~d ~ en~,t n~s primer pl~ while a 4~4-bp produce is ~mp~iflIet fr~m ~he wild qrpe alIele between pr~e~s pl a~d p3.
I~ne 2 is a repres~ntatlon show~:
(A~? growth ret~rded ~ A~L~a C~ c2 ~1- ~w born mAOUS~ (lef~ ~ar~ to nc~rmaI
C~ + / + litte~ 'natc (~ight) .
(B~ ~r ~et~,,~te mice at 3 wee~s of ~e {left) ~ ~ to a wi~.d type a~mal ~ri~ht).~!5 Th~ ~L~ L,,yiC effect va~ies ~ se~erity.

F;gUre 3a ~S a ~ AI Sh~ g mo~hoiOgy Of ~e cervical aCd u~per thOra~ic V~Vj~A_e in C~ ~1- h~ArO6r~,0lOS (Het~ a~d a wild type (WT) li~er m~te. The vertebral mlmber is sSlown on ~he left side of ~e pa~el. Vert~br~e 3t ~ a~d 7 and 10 firom heterazy~ous 30 anima~ sho~ molphol~ic~l ~eatures c~AIaractePAstic of the i.. ~Int~l~ cra~iial ~ertebrae in ~3--SE~ 16~ e~ Co l l ~sun C~.~e 61 :5 ~25~ Z~0 F'. 20 c~ 2184780 .

WT litoer ma~s control, This is best seen in expressio~ of a~terior tubercles in ~te~z ~v~,rle~e 7, abse~ce of these tube~le~ in ~lCt~v~-ygO~ G 6 and the pre~ence of a promînent spino~ process ~n hete~y~ e.~ ae 10. Abbreviations: PT, fora~en t~ansversum: TA, tu~erculi anterio~ PS, spinouspro~ess.
s Fi~ure 3b is a rc~r.,~e~ n showi~g malrv~ ion of the ribs. Sternum ~d ribs from three ~ +J- hetero~ygotes (2, 3 a~ 4~ and one wild type litter mate (1) showi~g an a~terior ho~ ; shift in the ll~,t~,.02~~ote animals. II~t~roG,y~,vt~ (2~ shows the eighth rib atlached to ~e ste~um on ~e left side. In ~ V~,~rgOl~; (3) the se~ond ~ib is a~tached to the 10 ste~um at the t~ of ~e ~ZIIII~iUl~J together wi~ dle first rib. The latter is incomplete and partiall~ atrq,hed to ~e se~ond rib (as is o*en seen i~ a ce~vical rib). The eigl~th rib is att~h~ to the ste~um bilaterally. H~ oLyg~ 4~ shows a similar arran~e.;npnf on ~he left, while t~e sec~nd rib has retalned its xlorrnal ste~nal ~ h~ l on ~e ri~t.

15 ~ 4 tS a ~ al;~ sho~ing tumours ~f the lar~ge iut~lin~ in C¢~2 +1- animals:
(a) gross appta-~ce of tumour at Ju~ction of cae~um and proximal c~lon (b) lower power section of preAvc~ ed tubular adenoma from prox~mal colon showing a~o~nlal c~ypt a,~ hit~ -c ~ to~cyli~ and Eosin (H&l~) stain.
(c) multiple mito6cs 1~ abnormal si~ ioJ~ cells from a tubulovill~ls 20 a~t ~ ~ the ~IVJI~lal CO10~1~ H&E st~n.
(d) ~ t ce~ a~d dysplastic epi~elial cells from a tubulovillous a~;n~a of ~e p colon. H~;~ stain.
(e) ...~ egion of ~cla~ elium wi hin a large bowel ad~ a. H&E stai~.
~fl tumour oells appe;~ to ~e penetra~ blo~ ~ esscl. ~I&;E st~.
2~ (~) absence of C~ s~in~g in the epithelial celL~ of a tubulovillous ade~lo~ from tbe p,~ al colon with ~lear s~aini~g of ~ .nt ~C)~ tissue.
~h~ sectionthr~ugha ~l~t~ tumou~. H~Estain.
(i) secti~n of small stlnP showi~g abnornaal ~illi ~t cryplS. H&E~ s~ain.

03-SEP-1396 16~ dlJ ies~ Co l l Ison C~ue 61 3 3-54 2770 P. 21 NC~ ~n6 2 1 8 4 7 8 0 E~MPLE I
GENE~TION O~ C~2 ~TANT ~ICE;

ES oell lines ~ere generated which ha~re one ~o~-f~c~iul~ allele of ~2 by corl~grl~iol~l S ge~e targeting. ~ G~2 target~ vector~ which r0placed most cf the first and all of the s.~bseq~,~..~l coding e~ons with a PGK-neo cassette was el~t~ dt~ into a W 9.5 emby~nic stem ~ cell line ~Figure 1). Nine C~2 ~uoc~u~ eell lines were is~l~ted ~ith a targeti~g err,~ of ~r~ ately 1%. Three of these cell li~es were selected for tbe gene~ation of chimeric mice by 701~51Oe~ Jection~ Two produced chimeric mice which transmit~ed 10 the mutation to ~heir plv~el~. H0~r.>~r~L~; ~,fLp~ were via~ole ~nd fertile, although ma~y had visible growth defec~ and ab~orm~l tails.

E~L13 2 G~;~OlYPI~ OF Cdz~ MUTANT ~ICE
G~.h~ Jiug of post~ J embryos at v~ious stages a~d of post-nataI a~imals showed that offsp~ing were ei~er ht,t~,~4~o~s or wild ~ able 2). Fur~ermo~e7 ~e ratio b~tween ~ese differed at bi~th from t~e eY~t~d Me~ n 2:1 ~ = 13.05 :~ ~0.01 whe~e df ~ 1). No post~ al deaths we~e recorded amo~g 181 liv~ births foilowed for 2g 20 days. r~ t~ly befare i~pt~n~*nn, llvmoLyg.)us n0gat~ve b~ b were dc.l~Grl~t~
by PCR a~d, al~ug~ numbcrs are small, el~ly~s approximate to ~e e~ 2:1 ~ati~
(Table 2). l~ese resul~s indicate ~at hO~OL~v~S ~ull m~ ts do not survive the pe~i-implantation period and ~hat ~e~e is pr~ably some ~,f~lemi~l loss of hctuoL~g~
emb~s durl~g gestation. Most of the he~ gous loss se~m to occur ~rly in gCs~ioll~
25 since an ~u~ ally high nu~ber of demons~atable ~ ion sites were not seen. Theea~liest eA~l~io~ of ~R ~Urin~ de~clop~,~ u~s in t~e u~h~t~rm at 3.5 ~ays; ~his may e~pla~n why null mu~t bla~ and p~ssibly some h(,~ g~tes fai~ to implant ~; r~s,~ully.

~s-SEP-139~ 16~ alJ ies c~ 1 1 is~n Ca~e 61 ~ 325~ 2770 P. 22 p~ ff 21 ~ 4 780 li~AMPLE 3 ~N~LYSIS 0~ C~2 MUT~NT MICE

C~2 b~,t~v~got~ show a range of ab~o ,n~lities. Many ~nimals ~re g~o~-th retarded at 5 birt~ and th~s is often associated with a sh~ or ki~ tail ~Figure 2). The ~ne is norma ly ~rw~ i~ the spongy ~ pho~lastic cells o~ the pl~cent~ (desce~ -nts ~f ~e u~ ) These oells co~ he $e"~ Iayer of the ~phoblast. Disturbance of dlc~ growth wo~d rcsult irl pl~r~n~l iQsurfi~ lead~n~ to fe~al deat~ or growth ret~ io~. F~Aan~ of h~ o~y2~uL~ place~tae at full ~rm i~iC~fe~ a relative paucity 10 uf ~he s~oJ~gioLrv~ho~l-ct~ layer il~ a number of spec~mens from ~rowt~ retarded fetuses Skeletal a~alysis of hctcro~,o~e ani~als showed an anterior l~oL,leo~ic shif~ uf the cervical ~nd ~horacic spinc in S of ~e 5 ani~s e~min~ (Fig~re 3a). Rib abnormalities, il~keep~ with tbis phen~y~ were also appareat ~F~ure 3b) Th~se results indicate that ~he 15 Cd~2 i5 hapl~suf~cicnt arld that Ioss vf function leads ~ an antP~rior ho~eoLi& shift. a ~i~u~iOJl similar to that d~i ibed for other homeobo~s m~tations E~1PLE 4 DE~3C~TION A~ ~AlY~S OF TU~IOURS
~0 F~s~ n of the g~ of ~0 h~,v~~o~ ani~S betwoe~ 12 we~ks and 28 weeks of age sl~owed mult~le adenomatv-us poIy~s ~y Ioca~d in the proximal large i~tes~ine of 18 out of 20 anim~ls. ~s~pi~lly de~hl~ tumours varied botweerl 1 and 10 in r~umber and we~ h~ ly present in ~e s~ll in~stine, as well as i~ ~e large i~ s~ r~ No tumours ~5 were seen in wild ~pe li~r ma~s of the he~ ~ J,.. teS. ~ ~s ~varied f~om those just ~isible to the nalced eye to ones up to ~ length ~nd 4 mm ~n width ~Figure 4a).
F~ , the largest were ~Ju u,~ (F~ , b). ~icr~;ic~l1y the 1arge bowel masses b~d the ~ of hlbulovillous ~dcnvm~. Crypt ~~ ue was grossly disnlrbed (~ig~re 4c~ y abno~mally situated mitoses were seen in dyspla~tic e~i~elial 30 oells ar~ giant cells were present ~ ures 4c, d). ~5 of m~t~l~Ci~ were ~so ~3-SEP-1~19~ Da~J le~ CQ~ On Ca~Je 61 3 g254 2770 P.23 cnv~ s6 218 4 7 8 0 -seen ~Fi~e 4e). So~c tumours ~d ~ be infiltra~g ~c mll~cul~-is mucosa a~d very occdsion~lly it ap~/cared as if blood vessels were pe~Lr~ Figure 4f). T~e epi~clium ~IIIlU~ll~ the tllmou~s w~ ~ 11Y ~b~ ,al~ showiu~ loss of muco~s sec~etin~ cellsin in~s~nal cryp~ an odle~ise gene~ally nomral arc-hite~lre W'hen colonic tumours 5 wer~ s~ined fol G~2, it was fou~d that the ~:;~elial cells ~ad cornpletely ~eased to express ~c gen~ in o~n~¢ast ~ clear C~2 stai~ e surrou~din~ epitbelium ~Fi~ure 4g). In t~e small imesti~e, m&l.)3~icaliy ~visible ~mc~s (Figu~e 4h) we~e less r~ but e~tensive regions in w~i~h ~e c~ypcs ~nd villi h~d a~ a~ea f~u~d ar~i~ttlre were ~ya~elll (Fi~ure 4i).
1~
EX~LE 5 FUN~TION OF Cdx2 IN MlCb:

~c~2 has multiplc ~ rl~Q~ . in ~e m~use, ~ludin~ a~ial p~tter~ f~lu~liol~ and the 15 l~ aLiUI~ of gr~ and d~t ~ testine. I~ the e~bryon~c L~u~l~n~s, h~G~ s loss o~ c~rw~ioll in ~e l,~p~ob~ast sto~s ~e process of i~ n"~ while k~ y~,O~Is ~dlion may inhibit this process or else lead to placentaJ i~C~lrr;~;ie E~MPLE 6 ll~MUNO~l~CHEMICAL PROOEDU:R~

Tissues were ~ocessed ~d analysed as de3e il~ by James et ~. (181. Tissue w~ xedin ~ e~ 609~ n~ 30~ w/Y chloroform, 10% vJv aceti~ ~cid~ for 1 h~ur at room ~lpera~ c, e~ ed~ af~ wax, ~nd CUt mtO S2CtiQI1S (3 ~m~. Polyclo~al 25 ~ ~;~dirs raised i~ rabb~;s again~t a bac~rially produced fusion prote~ the ~t~rmin~l 1~ am~no a¢ids of mur~n~ re tbe~ ~ed to ~etect the p~c~i~ in ~hesese~ions. Tbe ~ eil~ of the a.~ wa~ establis~od ~oth by Western blot a~alys3s ~8)and on tissue secti~S by ~ g tlle antisexa eit~e~ wi~ 5 ~ug of purifled fuslon pxotcin ((~ t~b~d~ng protei~ or t~e sa~e am~unt of ba~tetial p~otein (maltose-30 binding protei~). Anti~en~ il,~y cDmple~es we~e ~ic~lis~d with a p~l~u~id~ ~ased ~-SEP-13~16 16:1Z Da~Jies~ '-ollis~n Ca~Je 61 3 ~12~4 277~ P.2~
2 1 ~ ~ 7 8 0 ~s du~tion sys~exn and all sectio~s were cou~ltrr~tdm~d witb hf ~ ylin.

E~LE 7 DEYELOP~N'r OF ~ IICE

Embryol~c cells at the early Morella stage (i.e. ab~e 6-8 cells) f~om no~al mice are subje~t to electri~ redlLLcr~t to genera~e a tt;(l, 1a;d embryo. T~ese are ~rmined to pro~eed to the ~ias~ocyst s~ge. ~t t~e a~pro,~ tLme that the innct cclls begin to die or just prior to this time, -~- cel~s are infe~ed into the re~usi~ v~able e~t~a embryonic 10 membrane. 171e ~jeclion of ~e -1- cells effeetively replac~ the inner cell mass and -/-e.~ s co~l~ue to grow on a fui~ io~ placenta.

'rhe gellelaLivl~ of -J- mice provides a further useful m~dcl fo~ oe~ dev~loplue~.

15 Those slcilled in the ~ will apprecia~ ~at the ~1lve~on tescri~cd herei is susc~tlble to v~ and mo~ir~ ;nn~ OBc s~ rr~lly de6sribed. It i~ ~o be ~tL~h~d ~at thc inventio~ incl~des all such va~iatio~s and modif~lcations. The i~veDtio~ also ~ncludes al3 o~ the steps, fea~es, compositio~s and coll~ul~ds re~erred to or indicaled In this i,l,ocil~ Icn~ ivid~ally ~r ~ e(,tively, and a~y and all comb~atio~ of a~y ~w~ or ~ore 2~ of said steps or f~a~res.

~13-~EP-lgg6 16:13 D23~ie~ ~olllson C~e 61 3 g25l 2~70 P.25 r ~ NCFRV.~l~ 21~ 4 7 8 0 -2~ -TABLl~ 2 Genotyping ~f che offsprin~ frcim ~he Cdx2 heter~zygote ~n~elCrOSSeS

~ge No. of No. of N~. of tl+ +1 I;.tters l~ul,~iVlc~ ~mbryos typed 3.5 d.p.c. 4 0 34 10 17 7 4.5 d.p.c. 1 0 5 4 5.5 d.p.~. 2 0 11 11 0 6.5 d.p.c. ~i 0 1~ 6 10 0 7.5 d.p.c. 1 1 10 3 ~ 0 9.5d.p.c. 1 1 S 2 3 G
10.5 d.p.c. 3 1 27 8 1~ 0 11.5 d.p.~. 1 0 9 ~ 7 ~
1~.5d.p.c. 1 0 3 1 2 0 13.5 d.p.c. 1 3 4 1 3 0 15 14.5 d.p.c. 1 0 4 2 2 0 I6.5 d.p;c. 2 2 14 8 ~ 0 17,5d,p.c. 1 1 7 3 4 18.5 d.p.c. 1 0 7 3 4 0 Ne~ boJn 9 55 22 33 0 2~ 2~ days 32 181 82 99 0 Genotyping was p~rul~d by PCR e~cept for * w~ere the ~,~n~ was determ~ed by J~Ji~er~ t~ leSe a~liI~liS, it is n~t pos~i~}e to ~isti~guish wild type tll~ 5 f~om ~S ~L.v"y~o~s. Wild t~pe7 fl+, he~f~yA~t~, +~-, homo2ygote~ . d.p.c. d~ys post c~itum.

03-SEP-1996 16:13 ~a~J ies ~o i I isun Ca~ie 61 3 ~254 27~0 P. ~
y~ 2184780 BIBLIOGRAPH~;

1. Fearon, ~R, Vogels~ B, Ce~l ~1; 759-767, 199~.

2. McGL~nis, W et a~ Ce~ 283-30~. 199~.

3. James, R~ ~zer~ dci, .TJBiol ~em~ 32A6-32~1, 1991.

4. Duprey, P et a~ ~nes ~ DeYelop 2: 1647-1654, 1988.

~. GarnerJ ~ ~c Wright? C~ h~ech Lleve~op 43: 71-81, 1g~3.

6. hIacdona3d, P~ ~c Struhl, G Na~re 324: 537-54~, 1986.

7. Beckl F Erle~, T & ~a~s, ~ Dcve~op ~nam 204: 217-229, lg9S.

8. James, R, et al ~. B~ol. Chem. 26g: 1S237, 19~4.

03-SEP-13~6 1~ a~ie~ '-ollison C~e 61 3 9~54 2770 P.27 218~780 E~C. :. I~rANt:~v-~

S~U~SN~ LISTING

(1) GEN~RAL INFORMATION:
~i) AppLIcANT: BLCK, Felix (to be a~slgned t~ HOWA~P ~LOR~ 8TIlv-~: O~ 8X2BRIME.~TAL PHYSTOLOS,Y
c~r~~) ~ii) TIT~E OF I~VENTION: METHOP~; OF t~I~Gl~OSING AND TREATING
CA~ICER
iii) N~MBER OF S~1JS~ S: 6 ( iV) C~ ;S~ C~ DgESS;
(A) P~I~R~SS~ DAVI~S ~OI,LISON CAVE
~Bi STREET: 1 r '"rTLE ~oLLrNs ST~1~73T
( C ) ~ MELBOURNE
(D~ ST~ VICTORI~
E) COU~SRY: AUSTRF~IA
~F) ZIP: 3000 ~v~ ~OI;~Vl~h i~AnARLE; ~ORM.
(A~ I~EDIUM TX~E~: Floppy disk ~B) COII~U'~ M P~ co~patible ~C) OPE~TlNG 8YST~M I P~-~OS/MS-DOB
~D~ SOF~WARE: P~tontIrJ R~lea~e ~1Ø V~rDi~n Il1. a5 (vi) ~uKh~l APPLIC~ION DAT~:
(A) APPLICATI5N NUMBER:
tB) FILING DASE: o4-sEp-Igs6 ~Cj cLA~sIFIcA~oN

t~ii~: PRIOR APPLICATION DATA:
(~1 APPLICATION NU~ER: AU pro~i~iDnal; number to ~e assigned ta) PILINC DA~E: 04-SEP-lg96 ~V~ A1LOkN~/~CEN$ INFO~MA~ION:
IA) NAME: ~UCH~C DR, ~ J~N L
~3 ~ ~E~'~J~OCRET NUMBERI E~H~LM
(ix) ~ELE~C IJN1CATIO~ INFORM~TION:
~A) T~LEP~O~ 51 3 g254 2777 ~B~ TELEFAX. +~1 3 325~ 2770 03-SEP-1996 1~ a~ ies CGl l ison Ca~)e ~1 3 9254 2771ZI P ~
218g780 r;~ ANC~Y.47,~96 - 2g -~2) 1~PORMATION FOR SEQ I~ NO 1-( i ) 8~QU~NC~ CHARaCTERISTIC~
~A) LENGTH: g37 ba~e Pai~
~) ~P~ n~ e1C aCid t C) ST~DEDN~SS S~ng1e ~ TOPO~OGY: 1in~ar MOLECU~ p~ NA

~iX) FEATUR~:
A) N~ME t~ ~ C~S
~ ~ ~ LOCATIO~ 34 (Xi) ~EY~ ~ DESCRIPTION S~Q I~ NO: 1:
ATG q'AC CTC AGC l~AC CTT CTG ~C A~G GAC GTG AGC ATG TA~ CCT AVC ~ 9 Met TYr ~1 Ser TYr I~eU LeU A6F LYa A~P YA1 Ser Met l~r E'ro Ser 5 10 1~
TCC ~G CGC CAC TCC GGC G~C CT5 A;~C 6TG GCT CCG CAO AAC ~T GTC~ ~6 Ser Va1 P.rg Hi~ Ser GLY G1Y Le~2 Alsn L~u Al~ Prc Gln A~rl Ph~ V~1 20 25 3~
AGI' CCT CC~; CAG I~AC CC~; G~C ~AC GST CGT TA~ CAC GTG GCG GCC GCG 144 Ser PrO PrO G1n Tyr PrO AeP TYr ~1~ Cl~ Tyr E{i~ V~l Ala Ala Al~

GCG 5CT C~ AW CC~ AAC TTG GAÇ A6C GCT GAC TCC CCA GGG CCA T''C 19 Ala Ala Ala Th~ Ala Asrl r,eu Agp Se~ Ala Glh Ser Pro Gly P~o Ser T~C; C~C ACC GCG 'rAC GG~ GCC CCT C~ ~GC ~.-,AG GAÇ ~GC; AAT G~C TAÇ 2~0 T~p Pro Th~ Ala TYr G1~ Ala Pro ~eU Arg ~;lu A~p Trp Aa~ G;ly l~r 65 ~ 75 80 5CA C~C GGG G~;C: ~;CT GCG G'-A GC., AAC CCG GTA Ç;CC CAC taGT CTC A~T 2~8 Ala P~ Gly Gl~r Ala A1e. Ala Al;l AEin Ala Val Ala l~i~ Gl~ Leu A~n GGT ~GC TCC CC:G GCC ~;CC ;;CT ATG t~GC ~AC AGC A~;;C 6CC GCC GAA TAC 336 Gly Gly 9~r ?x~ Al;~ Ala Ala Me~ Gly Tyr Ser S~x P~o Ala Glu ~,~r ~ G0 ~os 110 ~3-SEP-1~l~6 16:14 Da-/ies Collison Ca~)e ~1 3 ~254 ~,7'0 P.:~

~or ~ ;CPRv ~6 - 3~ -;:AC 5CG CAC CAT CAC CCG CAI~ CAT CAC CC~: CAC CAT CCG GCC GCC TCG 3 t4 A1a H1~ H1~ ~{Ln Pro E~l~ Hi5 H16 PrO ~i6 ~i6 Pro Ala A1a S~X
115 1aO 125 CCC; TCC TGC GCC TCC GOC TTa CTC CAG ACG C~C AAC CTC GGG CCC CC~ 43 PrC~ Ser CY~ A1a Ser G1Y ~eU LeU G12~ Thr ~eU A~n Leu Cly E~ro Pro 130 135 1iO
GGG CCC ~CA GCC ACC GCC GCC GCC GAA CAG CTG TCC CCC AGC &5C ~AG 4 ~ O
Cly ~ro Al~ Al~ Thr A1a ~ . G1U G~n ~eU SOr ~0 S~X G1Y a1n CGG CGA AAC CTG TGC GAG mLGG AT5 CGG AAG CCC GCG CAG CAV TCC CT~ 528 Ar9 ArSr A3n LeU CY~ G1U TrP Met Arg LY~ PrO A1~ G1n G1~ ~er ~ eU
~65 170 175 G5A AGC CAA 5~G AAA ACC AGG ACA A~A GAC AAA TAC CCC ~T~; GT5 TAC 576 G1r Ser 51n Va1 LY~ Thr ~rg Thr ~Y~ ~P LY~ TY~ A~g t,tal ~J~l TYr hCA ~;;AC CAT CAG CC~ GT~;: GA~ CT6 GAG P~G GAG ~rT CAC TTI AGT CG~ 524 Thr A~P Hi~ G1n Arg LeU G1U LeU 51U IJY5 Glu Phe ~i~ Phe Se~ A~g TP~C ATC ACC ATC AGG A~V AAA AGT ¢Z~G CTG GC'r ~3CC ACA C$T C;5G CTt~ ~ 72 TYr r1e ~hr I1Y Arg Ar~3 Ly4~ Ser G1~ Le~1 Ala Ala Thr L~u G1Y LeU

TGC GAG AGG ~AG STG AAA ATT T~G rrT CAG AAC C5C A5A ~::C AAÇ GAG 720 Ser Glu Arg ~:ln Y~l Lys Ile Trp PhQ :;ln A~n Arg Asg Ala Lys GlU
2aS 230 235 24i0 AGS AAA ATC AAG AAG AAG CAV CAG CAG CAA Cl~,G CAG CAG CA~: CAA CAA 76 c Arg ~y~ 2~ ~ yf~ Lyf~ Lyr3 G~ ln Gln Gln Gln Gln Gln Gln Gln &ln CA~; CCT CCA C~& CCG CCG CCA CAA CCT TCC C~AG ~CT CAG CCG GGT GCC 815 Gln Pro Pr~ Gln Pro Pro Pro Gln Pro Ser Glll Pro Cln Pro Gly Al~
2~0 265 ~7~
CTG CGG AGC GTG CC~ 13AG CCC TTG .~GT S~CT GTG ACC TC~ TTG CAA GGC 864 Leu Arg Ser 'Jal Pro Glu Pro ~eU Ser Pro Val Thr Ser Leu Gln Gly z~s 280 285 IZ13-SEP-l~g6 10: 14 D~IJ le CG l l ison Caue 61 3 ~J254 ~70 P. 30 F.~ ;c~ 4nl9G 218 4 7 8 0 T~A GT~: CC'r C~ scr GI~C CC~ GG~ G~ Cl'G GGG CCA CCT ~A ~;C; GTT ~12 Ser V~1 Pr~ C;ly S~r ~ral Px~ Gly Val ~c~u ~ly Pro Ala Gly Gly ~1 295 ~5 300 TTA ~ ~CC AC~ G~C ACC CAG TGA 93 7 Leu A3n Ser ~hr Val Thr Cln 305 ~10 2 ~ ~FO2MATION POR 8~ NO ~ 2:
(ij S~QUE~CE CH~ TI3R1S~ICS:
~A) LE~T~i: 311 Rmin~ aCidg (B~ 'rYP2~ am nO ~Oid ~D~ TOPO~ nV~
~ii) MOLECULI3 T'YPE: prctein (Xi~ S~;~Ur1~ BSCE~P1'IO~I: SEQ ID NO:~:
Met Tyr t~al Se~ Tyr Le~ Leu A#p l~r6 ASP Va1 Ser M0t Tyr Pro Ser Ser V~1 Ar~ Hi~ Ser G1Y G1Y L~U A~n ~eU A1~ Pr~ Gln A~n Phe 1~d1 Ser Pxo Pro Gln TYr PrO .~8p TY~ ~ Y ~1Y TYr Hi~ VA1 A1a A1a A1a A1a A1a A1~ 'rhr A1a A~n Leu Aap SF3r Ala Gl~ Ser Prc~ Gly Pro S-r S~ 55 ~0 Trp Pro ThX ~ yr Gly Ala P~o L~u Arg Glu Aup Trp Aan G1Y TYr 70 75 ~
A1a Pro ~ly Gly Ala ~1~ A1~ A1a A~n A16I Va1 A1a ~ Cly Leu A6n ~5 90 95 G1Y ~1Y Ser PrO A~ A1a A1a Met Gly Tyr Ser Ser Pro Al~ ~:lu Tyr 100 105 11a t~16 A1a }li8 H1~ Ni~ P~C~ H~ XO li~ PrO PA1A A1~ Se~
115 1e;0 125 Pro Ser CY~S A1a Se~ G1Y ~QU ~e~ ;i;lsl Th~ L~U A~rl Leu G1Y PrO PrO
~3~ 135 14C

~3-SEP-1~9~ 15 I:au i~ Co l l lson Cave 61 ~ g254 ~7''13 P. 31 D ~ V ~ ~~/Ç6 2 1 8 4 7 8 0 Gly PXO Ala Ala Thr Al~ Al~ Al~ Glu Gln Letl ~;er Pro S~ 51y Gln 145 15~ lSS 15C
Arg Arg A~h Leu Cys ~;:lU Trp Mct Arg ~ye Pro Ala Gln ~ln ~er Le 16S .7~ 175 Gl y Ser Gln V~ï LYEI Thr ~r3 Thr Ly~ ~.p Ly~ Tyr Ar~ ~A V~l Tyr 1eO 1~5 190 ~hr ~ap iiic~ AI-g L~u G1u t.et~ ~1U I.y4 C;lu Phe E~i~ Ph~ Ser A~g 1~5 ~00 2~5 I'yr Ile ~hr Ilç ~rg Arg Lya ~;er ~,lu I,eu Ala P.~ r ~eu Gly Leu 21~ ~15 ~20 Ser Clu Arg ~:ln Val Lye Ile Trp Phe Gln A6n Arg Ar~ Ala Ly~ Glu 225 ~ 0 23 5 2~0 Ar$~ I.y~ ~le Ly~ ~y~ ~.y~ ~lh ~;:ln ~lr. G11 t:ln 51n Gln Gln Gln Glh ~ln Pro Pro Gln Pro Prv P~o Gln P~o Sçr ~:ln E~ro ~;ln Pro ~ Al~
26~ 265 ~70 L~ S~r Val Pro Glu Pro Lsu S~r Pro Val Thr ~er Lou Clln Oiy ~7S 2~C 2~
Ser VAl Pro Gly Ser Val Pro Gly V~l ~eu Gly P~o Al~ Gly C~y V~l I,eu A~n Ser Thr VR1 ~hr Gln 30~ 310 03-SEP-19~6 16:15 r~av ies 00 l l ison Ca~e 61 3 ~254 2770 P. 32 ~2) ;~PORMATIO~ FO~ S~Q I~ NO:3:

tA) ;~ G~H 2S bA~e pAirs ~B) ~YPE: hucle~ c llcid ~r) STRp~ '5s: ~ingle tD) TOPOLOGY, lin~ar MOLECULE TYPE: oligonuc~l~Qtid~
~xi) ~i~yu~ ESCRIPTION: S13Q I~? NO:~:

T~GI'~ 5.~ L~;dG AATCC 2b 1 2 ~ IIiPORMATION F0R 5!3Q ID ~O : ~:
~A~ L~NG~ 23 ba~ p~ir~
1~ TYP73: nucleic acid ~C) STRA~D~DN~SS: ,~in~le ~D~ ~C1POLOGY: linear lii) MOLECtJLE TYPE: Oligo~u~l~o~ide ~xi ~ S~;Qv~N~ )ESCRIPTION: ~BQ ID N0: 4:
ATA. ~ A A~;A~C~SGCC GGC ~3 03-SEP-l~g6 1~;: 15 rl~ i es Cc I I i son Ca~ e 61 3 925~ 2770 P . 33 2184'780 r ~;~ FRV 4JS~6 (c ) IltFORMATION FOR SE2 ID NO: 5:
(A1 II~GSH~ Z2 ba~e p~ir~
~8~ TYPE ~ nu~!ie~ ~cid t~1 3TR~Er~NR~q5 ~ingle ~D) TOPO~O~Y. linear (ii1 MOLI~¢V~ TYPE: Oligonuc'e~t~e (Xi~ J~rJc~ ESC~IPTION: S~Q ~D ~ 5:
GCGAC'rA2TC ~C~AGAGG A~i; 2 2 t 2 ) INPoRMATIo~ FOR SE~ NO: 6:

ti~ V~ HARACTE~8TI~S:
(A~ ;l~': 1162 ba6e p~ir~
YPE: nu~:leic ac~ d ~C) STl~U ' ~ ~hlr~S~ ngle ~r~) TOPOLOGY: IinQa2~
14L13CVIJE TYP~: ~N~ (gencmic) ~xi~ SEQV13:NCE D~CRIPTION: S~Q ID NO:6~
A'rCCCAGC CA'rccACT~A TTAcTt;:Gt:TT CcATATc~G GGTC'C1~5TTG l'TGGCTTTGh 6 0 AGAt;G:GTTTC GAT'r5~TTAi~ TG~AATAGTT l~TAAAAATG AAllGCll~C CTTGAACTTA. l20 ~,ATC:rTAAC~ GTAAGAC C~ GG~C&CAA CCTGGAGGTA GAA~80 TAGAGGCCGA CTGG m C~T TSG~ GC~ AATAAATGTA ~T~CTGA~GG 24~
~ CC~hCC GlCC~lCC~l CA~ CTGGTTCTCA GAGC5CGA~G GAA~AG~CT~ 300 CACCGCAGCS CTACAA~CA CC~ACCTCC~ ~lCA GAAAGCTCTT CA~AGcAA~G 36~

A~ LC'CCC CCC~CCG~ CCCCC~ TGACCACCTT Cr~CCTGAG~ ATGT~C~hAA 420 CG~.GCG~C GCC~1LC~ ~AACCCAGCT TCC~ACCAAS C~GCC~lC TG~ACTTCCC 480 AAGGCGITTG CAA~l~ CATTTCAGTC TTTGAACC~G TGATT5GA~C- TTAAAGTGCA 540 03-SEP-l~ Da~ies Colli~on CalJe ~1 3 ~254 277~ P.3~

c.n.Y.~
-CCCAG5TIGG AAGCAGGAAG GTGGTAGCTA CC~AG~AGGG Cc~GAATATT CAGCTCT~ C0~
C~GGTCGC~G ~CC~ ~GG A5CGCCCCCC ~CC~CAACTT TTAAAATGCA AAlC~LGlll 660 CTcGGGcT~ TG5T~5~GGG GACTGTGTGC GT~GCGCGCT GCb~-~lCGA~ GT~TCC~GCC ?20 A~l~alUl~ GTGTC~TTAC TAATAGAGTC TTGTAA~GAC TCGTTAATCA CGTAAGGCCG 7~
~lGG~-L'GGG ACTCC5CGAG CCAA~CTGCG ~CGGGTCATC ~CCGCCTCTA CAGCTTACTG ~40 5~5~GGT WGAÇ~AAAG AAGG~ACAG~ GGGGA5GAG¢ CAGGACGSAC C~C~CTG ~00 CCCGGGAGGC ACAAGC~CTG ~AGGAGCCG A~GGAGCACC GTGGGCTGAG GTGCAGCCAG 960 C~ACC m AT CSCTAGCCSC C~GCGCCTCC C~C~vlC~ CC~CAAC bl~ CCCC 1020 AGACACCATG GTGAccTcTG ~.~lG5~C C~CGCCACCA SGTACGTGhC CTAC~T~SG 1080 GACAAGGACG T~GcArGTA SCC~AGCT5C C~G~ACT CC~G~C~ GAACCl~G~ 0 CCG~r-~CT T~GTCA5TCC TCC~CAGTAC CCGCA~T~CG G~GGTTACCA CGTGGC~GCC 1200 GCGGC65~G CTAC~GC~AA CTTGGACAGC GCTC~CTCCC CACC~CCATC ~vlG~CC~CG ~260 GCGTAcaGc¢ CCCCl_iCCC CGAGGAcTcc AATGGCTACG ~ACCCG5~G CGCTGCGaCA 1320 GCCAACG~G~ TAGCCCACGa T~SCAATGCT GGC~CCCC~ G~ AT CGGC~ACAGC 1380 AG~CCCCCC¢ A~TACCAC~C GCACCATCAC CCGC~TC'A~C ACCC~CACCA LCCG~C~CC 1440 ~C'GC~lCC~ GCGC5TC'CGG ~ AG AC5C~CAACC ~G~GCCCCCC CGGGCCC5CA 1500 GCCACCGCCG CCGCC.5AA~ ~lCCCCC AGGG¢CCAGC GGCGAAACC~ GT~C5AG~5C 1560 AT5cGeA~cc Cc~c~cAGcA 5TCCCTA5GA AGCCAA~CTA A5C5~1~CC~ GGGGGCGC~C 1~20 GAGÇ1~CGC ~;~CCr~11GG~ G~ G1C~G~G G~V~L~1GCC ~C;C~CCTACC CA5GACAGGA 1680 GAA~G~,CAA A~GGGGAAAT AcA5G~GGAl TC~TCCCAGG AGT~TATATT ~ CC 1740 TCCCCCACG11 TTG:~rrAGGG CAGTACC"CCA ~GGCAACCT CC,GGACGCCT 'rC(;,GAAGCTl: 1800 CC~G~AGT~T CCTAA~C~r ~CGCCCGCC' GCGAGCTG~A ~ CC'~1~ GA~ G~C1~ 1~60 CCLC~ AT ~GCrTACACC ~ ;!r a~c5 CAGA~;TCG GGCCGCT~Ga A~C~C111~ 1~20 ~3-SEP-1g96 16:16 D~ies Collison Cave 61 3 9254 Z~70 P.35 C~Gl~CCl~C TCAACTTAGC TACCTGCTTT TCl r~, J ~A TCTGAGGTAC CCCCCCCCAA 1350 AcGccA5~GA GTGTGGAGGc TC~GCCAGGA SCAGTTTGGC CATSAGGGTC TACTG~GG~ 2040 GT~G~lG~ CTGcGA~cAc AGACGCCCCG G~GCGCCCCT G~C~C'AA~G CGGAGCC'CCC 2100 AG~AGCCTCG CA~GAAGT5G GGGAACTCCA ~G¢~CC~ GA ~lCl~GC~lC SCICTGCCAA 21~0 CC~AAGTC~C TCATAGAA~ CCTCTCTTAG CATCACAGCG ~ACTCG5CTT CTTAACAAG~ 2220 ~ ClG ACCCllC~l TCTAGAATCT CC~GTGTCTG AGA~S~ITGT ~TGTTGGA~T 22ao C¢~ ~TT ~ICPCG~ ACCCGAGATT ~CCG~ GÇGACSCACA AG~ L 23~ 0 GcccTTTcTG Al~ GAT ~TCT~TAGGA GTGGGAAGGA AGTG~GCAAA GTGTCACAG5 2400 GAGTCG~ACT M GAGTCGCC C~ T~l ~ hGC~ C A llOA
G~I~CÇ~l~ CTcs~GATTT ~ GG~ACAGCCA GGCCACA~GC CCGGCLrA~G 2520 GccAcTT~GA G5A~GTT~AT AÇCCAATTAC h~CCG~ATAA ATCAGAGCCC C~Gll~GGGG 25~0 CC~uTCGCTG GACACTTCTG CGTCACTGCG CAGACCCCCT CAACATCTTG ~T~GGCAATC ~gC
~lCCÇC~AGT G~AGSCTCCA GATTAaCCTC CA~-o~ L TGAcATTGTc ArTTCCAGGA 2700 GGACTTACTC CTA~CGG~T TG~CCGCAT~ A~CCCTACTT ~CCCAA5C~T GCCTGAAAGG 2760 TA~GAGCCÇ AGGAT~GCCG GTAÇ~CG~CT TA~CC~lC~G ~AAGAACTTT T~CGTTCA~G 2820 CCC1~,~G TCCGAGACTA GG~1C~G~C AGGGGGAGGA GAACCTCAGG AGCCC~4CC 2~AO
~4~l~AG CAGATGAGAC GT~G~ ~A AGTCCCGC5C CG~GGIGGCA ~GACCG~l~l 2940 CçGcGccGGG C~CC~L~5 ATCS~AATvT AGGGA~GCTG l~GCGC~CAG IGCCGGA5C~ 3000 C~AC~ CCACA~ ATCT~SCAAG ~G~ CC GTGTACGCCG C~CC~cc 30~O
TTTGA~GTAC ACCT-TTCAGC CCTGCCATTG L~ lAG 6GCCCGG~AA ~AG~v~ 3120 ~ CAG Tc~ATAATcA ATTCTGACAA CTC~ A IC~Ci~CC~l C~CCAGCCC 3180 CC~ l.G5 T~CCCAGGGG GSGCGGG¢~C CCT~C~AGCC ~L~a~A~C GGA~CC~GG 32~0 GCTC'ACGGAC CAI~L~ T~CC~CAGCC AGCGC~lGCL GC~CTA~GAG CAGCATCCGS 3300 03-SEP-1996 16:17 r~uie~ Collison C~e 6, 3 9254 ~770 P.36 t ~ NC PI~

TCTAGaGCG~ GGACC5CTGT CCC~A5~VT l ~ l G rvGcG l C C;C~TCCCAGG TCTGATAGTT 3 3 6 0 TTCTTTACTA CIACA~GC'TC TTTGCTATTC GG;TcTccAcA CTTAAGATTT G~~ C';: 3~s20 TCl1C'~G A(,l~G;GCAC TAGCGACTGT GrrACAGG~T CCAGTGAGCC CG~ .C~.GAC 34 CGGTTG~ATC CCTT~rAC~C CAAAGCAGG:Ç ~:;C~GÇ~ G 5GGc~;cAGc~3 CCCATTCA~ 3 S~ 8 l~GivGC~ lGCt;; GGGClG-'ill TATG~AATG L" ~.L~GCGG GGA~GGGC;P~T GAAAGCT~:AG 360 Ç~CCGCCATn' GCI'CAGTAGT GC;TAA'rTGAA .~,CA~AATGi::G CTTGTTAI~A AGC;CATTGAA 3 ~ 5 ~
AGCG~ C TTTGAT~CA ~ TTGCATTGTr TG~GACT5TG TTC:CCCTrrC 3 ~2 0 çAcG~iGCGGG GGGACCTt:TG TCTGATCCÇT C:CTGTATCTT CC~GGP,CAC TTTTGTAGTG ~780 CTTTGÇAAAG GTTrr~ACCT GAACAAAGTC AG~ GG~7 GCCTTA~A~A CC~CTG~GCA 3~4 ~CT~CCACCC ÇTTAGA~CTC C-~ 1 Gl ~AAC TA~4~ACT~A ~AC AAAAT CTA1C~'~L~1 3gO~
TGAT~TG~GA CC1~1C~AAC TTT¢Ç~CG~5 GAACAGTrCC TGGCCAGCAT T~ CCaTT 3960 CA~GCATCCC CGACr~AACC GGGTT0AAT~ 5CCAGACTCC ~GCCTGATCC ~GCAGCCCCG 402 CGCTATTTGT CA~GCTCGTT C~GTAAAACA ATCAATCCAA GA~TTAA~AT ~TT~C~C M 40 TCCAATGATC ACAGGTCACIr T~G~CAGGAT A~L~1GG GGGGATAAA~ GGX WhATTAA 414~
AAAAGAh~GA ATGCCCCAGG CCAh~CCTAC CTGGGCAGGT GAG~CGCGT. CCAAGTAGAA 42~0 ~ 111C~1 CC~5ACAAC~ T~AGA~AGC ACTC~G~ATT CChGGGCAG5 AC~ 4~60 'r6GTACTGG'r GAC~ ~G GATCT6CCAG CCCA~g::A'rC CA~ lCCA CTATATTGAA 4320 AC~AnGGTA AC~ACCCGCT TAC~C~GAC5 CC~CA~CCCA GGIATTTrCA RA~aBCGLG 4~0 ~TGTT~CATT TCrAA~A~a~ CT¢A~ATGT GGATATT~AT Aq-C~-GÇGG A~C~ A ~4gO
~-i' L l~CCACC CC~C~llll~ C~GATCC~A ATAT~CrATA TTT5ATTTAT 6CA~AGGACC 45~
G5GG~ 5L G~GAGG~GTT CCTTTCA~GT TAGITGTGC~ T~GA5GCrTA CAGC~CCCTT 4550 ~CCCA¢~ATC Cl~l~G~5~ ~ATTGTCAA CCT6GGTA~T AGACACCCCC CTCCA~CAG~ 4~20 ~5ATGGAGAG M ATGACTCC TCGCTTA~CG A~ ~A TSACCTATGA CTGA~CGATT 4680 03-SE~-1g96 16: 17 rlav I es Co I I i so~ CA4 ~ 61 3 gZ5a, Z770 P . 37 218~780 ~..V ~ ,~I~,tllY 4~n6 GTAGTTI~ OCCAAAACTT ''T.. ~ ~CC1C C,2~C~GT6AAA AÇCAG~ACA~ A~ TA :1 7~0 C~GTGGT~: TAC~ACAGACC A~C~S~CGGCT GGAGCTG<~AG A~GGAGTTTC ACTT'rAGTCG 4~0~
ATACATC~CC ATCAGGA~GA AAA5Tt;AC;~T GGCI~;CC.ZCA C1 1~;~GL~ CCGAGACGC~ i~60 GGTGGGAATC ~G~ CL~ G ~ 1A 'rTAT ;GCC- G GGGCAATCIT AATC~GTACC ~92O

Al'~AqAF~ GG~Ct~AGG CAGCT~;TSGC ~TTT5A~ TC5TTGAGTG T:~AGT~:T~ 4980 TTTGCCG~Tr TTTCA~TG;~A ~r~P~r~ T ~TP,AGGG~L~ P.6CCAAGTCG AGGCTA'-'AGG 5040 ~;~GGAAC~5~ ACTTGC;~CAC; ~(~AAAGAGC3 ArrGG~CCA T5~ 1 ChA C~ GGG 1~ S1OO
GCGACCCCSC AGCTTCCCAC TCAAACA6GC TG1GCI~'1AT AATA~C~GCA A~TTP~TA~T 51F~Q

TATGAAATA5 CAATTA~A'r AATTTG~I:;A~ TGG~ ,' ACCAC~ACA~ GAAGAACTGT 52 2 ~
C~ AAAAGA A~"~ CCA:;TCTAC GCATAC~l.C TAAAG~rCT¢C TCA~rACAAAT 5280 T~A5AGGCA GGG~GC1C;C~,~; A~T5AT~;:CT TF,AGA,hCACA CAC1~_11G~.. A GAWATC5Ç~A 5340 ~Ll~ , CAGCA'rCCAC~ C,TCAC;GTGGC 'rCACAGT~ ACAGTC'rrGG 5~TAACTCTGA 540~
~GCAt;~ ;A CAC. C~C5 CTCTCGCTTC T~:TGGATA~ ~rATGAATSTA TACACDCAT~ 5460 CACATATCAA At;AT'r~A~AA A'rCCTI 5~AC ~ 1 C A5~A~C~CAA AAGAG~CCCT S 52 0ACTTGC:ACAC CAP~CACTTTC~ r~TACTcTAG GÇI~,AGGGA AGGAACAAAG ~GAGAAA_T 5580 cA'rA~CCA~ G~ 1WA CP;~AeJm.~. ~ CL~C CA~,GC~GP.AG P;~ TCGCAG S640 TACCAATGA~ <;~;;AGC CCA~GGCTGA CCCACGAG:C;C ACA~ J:CA5CT G~';AACCA~ 5 7 rJ rJ
T~TGTAGGÇ AAmLAGCSTTC C~:ATGGTTC ~11CC~ 1GG TTCTGA~:;TT ~ 1hC 5760 T~AGGCTT~ 1 1 16r~ L .L 1 il TTTI TGAAAG CAAC~T&G~ C AG~CGGCAGC S 8 2 0 ~AA~TP~AAGA ~;5PCGA~CT ÇCCC~ 1 TWGA~CATT TCCCAAAC~C AGTGAAA5~A 5~8 GCA~GC1CCC G'GGGTG~COC CA1L1~ C TrC1"rATC~TT CTT~T~ 'rT CTAI~11G1G 5g40 A'rC'rCTAG5T m~AAAA~'rGG IITCA5A~CC GCA~;AGCCA~ 3AGAG5AAA ATCI~AGAAGA 6000 AGCA/:;CAGCA 5CAACAC~AG CAGC~CAAC AI~CA~3CC'rCC AC~GCCGCCG CCACAAt: C~rr 6 0~ O

03-SEP-1~96 16:18 Da~ies Collison Ca~e 61 3 ~254 2770 P.38 21~4780 }.'.~ RV . ~

CCCAGCCTCA GCCGGGTGCC CTccG~AGc~ TGCCvGAGCC CTTGA&TCCT ¢TGACCTCCT 6120 TaCAA~GCIC Avl~ ~vl ~ CC~TG G~u~ G GCGAGCTGGA G~ lAA 6~80 AC~CCACTGS CACCGACT5A CCCCTCCCGT GGTCTGAh~C GGCGG~CGCA CAGC~ATCCC 52~0 AGGCTGAGCC ATGAGGAOTA T~GACGCTCC GAGAATCCTC A~C~r~TT C~lClC~lCC 6300 TACCCACGA~ CAGCA~CTAC TGATGGA5AT TGAGGACAGA AGATGAGTGG A~TTASGGAC 6~60 CTCAGGCCAA CAC~TG5TTT AGAlll~ 1L~A A'l~ CA TTCCGACTCT 6420 T~CTGCCAGC ~A~n~ AC CA~G~G~TTC ~GG~ ~AT5 ~ L~C~A 6~80 WAGS~A~TA CC5ACATGAA ~CTTTCAG~C ~ G CA~CTC m G C~ -Gl~ 6540 l~ GA~SIGAG~A GAGAGTGAGA ~TG~vATG~ 1~GGv~AGC AATACTTGA~ 6600 CCAAv~TGGC r~ C~l~C TGACTGCTTT CTG~GAACCA G~lG~jCC~ C~G~lCC~G 6660 GC~AG~G~CT ATTC~AACTA ~A W ~GCCAG ~GGCAGCTAA GAThGCTG~A ~T~jACCGAA5 6720 TCTGCA~AAC ~-CCCCC~CC A~l~G~-G GG~ Cll~ T~CACAAATC AG~AAÇGG~T 6780 GGlGGj~.~A ~eGw GCGG ~GAG~GG5GG TTGGTTAGCC AACCCCAG~C CCCTGCCACA 6840 AGGC~ T~AAA W CT GT~ACCACA5 C1~21~lAG5 CGGAAT~AT Gl~ G 6900 ~AAATvCCAe AGCCAACCTG GA~ LGl' ~CM lC~Ç G~ lGG~l ~AAGAAaAcc ~960 ~6AATTG~T GC~5CTGT~C ~AGTCACT6A .~ lA~C GAGCCAAAC~ A~ AA 7020 AAAGCCTTCT TGATCCATGG GTAG~GA~GT T~TATGGTCA AGG~ TCC GGAGGGGG~G ?~80 AA¢GGGATCC -~CA~-TT GACTT~TATT rTGSAAAkA~ ACAA~GATA~ AC~AACTTrA 7140 ~ChGAA~AAA A~AA~AA~ AA ?162

Claims (19)

1. A genetically altered animal or progeny of said animal having a predisposition to develop growth of neoplastic cells in intestinal epithelium.

2. A genetically altered animal or progeny of said animal according to claim 1 wherein said animal comprises a mutation in at least one allele of a homologue of the Drosophila caudal gene.

3. A genetically altered animal or progeny of said animal according to claim 2 wherein said animal comprises a mutation in at least one allele of Cdx2 or equivalent thereof and which is a homologue of the Drosophila caudal gene and wherein said genetically altered animal has a predisposition to develop growth of neoplastic cells in intestinal epithelium.

4. An animal model for carcinoma of the colon, or a precursor stage thereof, said animal model comprising an animal or its progeny having a predisposition to develop growth of neoplastic cells in intestinal epithelium.

5. An animal model for carcinoma of the colon or a precursor stage thereof, according to claim 4 wherein said animal comprises a mutation in at least one allele of the Drosophila caudal gene.

6. An animal model for carcinoma of the colon or a precursor stage thereof, according to claim 5 wherein said animal comprises a mutation in at least one allele of Cdx2 or equivalent thereof and which is a homologue of the Drosophila caudal gene and wherein said animal has a predisposition to develop growth of neoplastic cells in intestinal epithelium.

7. An animal model for carcinoma of the colon or a precursor stage thereof, according to claim 6 said animal model comprising a mouse or its progeny having a mutation in at least one allele of the Cdx2 gene or its equivalent and which is a homologue of a Drosophila caudal gene.

8. A method of determining a subject's predisposition to developing familial carcinoma of the colon, said method comprising obtaining a biological sample from said subject containing cells and screening for the presence of a mutation in a homologue of the Drosophila caudal gene wherein the presence of a mutation in at least one allele is indicative that the subject is likely to develop carcinoma of the colon.

9. A method according to claim 8 wherein the gene to be screened is Cdx2 or its equivalent.

10. A method of diagnosing colon cancer or a likelihood of developing colon cancer in a subject, such as a human subject, said method comprising obtaining a sample such as a biopsy sample or a sample obtained following surgery of the subject's intestine and screening for the presence of a mutation in an allele of a homologue of a Drosophila caudal gene such as a Cdx2 wherein the presence of a mutation in at least one allele is indicative that colon cancer has or will develop.

11. An antibody to all or part of Cdx2 for use in screening for the presence or absence of Cdx2 expression.

12. A method for detecting Cdx2 in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for Cdx2 or its derivatives or homologues for a time and under conditions sufficient for an antibody-Cdx2 complex to form, and then detecting said complex.

13. An isolated nucleic acid molecule corresponding to a human homologue of the Drosophila caudal gene, Cdx2.

14. A nucleic acid molecule which encodes human Cdx2 and comprises a sequence ofnucleolides having at least about 60% similarity to the nucleotide sequence set forth in SEQ
ID NO:1 and which is capable of hybridising under low stringency conditions at 42°C to the nucleotide sequence set forth in SEQ ID NO:1.

15. A method for cloning a nucleic acid molecule encoding human Cdx2 or a derivative thereof or a part thereof, said method comprising screening a nucleic acid library with mouse Cdx2 DNA or mRNA or an oligonucleotide probe based on mouse Cdx2 DNA or mRNA.

16. A method for cloning a nucleotide sequence encoding a human Cdx2, said method comprising searching a nucleotide data base for a sequence which encodes a molecule having at least 60% amino acid similarity mouse Cdx2 or where the nucleotide sequence is at least 60% similar to the mouse Cdx2 gene, designing one or more oligonucleotide primers based on a nucleotide sequence located in the search, screening a nucleic acid library with said one or more oligonucleotides and obtaining a clone therefrom which encodes said human Cdx2 or part thereof.

17. An isolated human Cdx2 protein including a recombinant form thereof having an amino acid sequence of at least 60% similarity to the amino acid sequence set forth in SEQ
ID NO:2.

18. A method for modulating expression of Cdx2 in a human, said method comprising contacting the Cdx2 gene according Cdx2 with an effeCtive amount of a modulator of Cdx2 expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of Cdx2.

19. A method of modulating activity of Cdx2 in a human, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease Cdx2 activity.