CA2022670A1 - Human tumor cells implanted in non-human animals - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A non-human animal model is provided with human cancer cells supported on a support including a biological response modifier, with the animal preferably being immunodeficient with respect to its own immune system. The animal model can also be provided with a human immune system. The model can be used for testing cancer treatment for primary human cancer cells.

Description

2~22g70 !

I)W4JaENE003 HUMAN TUMO~ OELLS IMPLANTED lN ~ON-HUMU~ ANIM~LS
Thl~ invontlon relat~ to t~mori~en~ and more par~icularly to malntainlng ~nd t~tln~ h~6n c~ncer cQll~ in non-hu~n animal~.
Wh~ ome tumos cell~ cAn be r~adlly ~rown in cultuxe, th~y de not, in ~h~ nvironmont of the ti~u~ culturo ve~sel, maint6in thQ it~nt~cal propertie~ o~ the prlm~ry tumor. In ~ct, th~y may ~req~en~ly repre4~nt a subtyp~ or aeloctet ~ubpopul~tion of the primary tumor. Althou~h t~
prop~rt~ of continuou-, and non-contact lnhlbitet ~rowth may par~l~t, numerou~ other charactarl~ics, including ~h~ ~en-s ~pr~sed, the na~uro and ~uantity o~ tho marker~ oxpr~ d on th- coll sur~co, and tho re~pon~a to hormoneo, b~olo~ic~l re~pon~e modiflex~ ~nd ot~r growth factors m~y b~
hi~hly varlabls, an~ dif~rent Prom the prlm~ry tumor of origin.
Th~e oxi~t c~rta~n anlmsl mod~ for th~
m~intbn~nc~ ~nd propa~ation o~ t~mor c~ . In mice, for examplo, ~umor call ~ot~l# exiEt, wh-~o lnJ~ction or transplflnt~tion og c-118 will r~Bult ln t~mor devolopment. HOWQV~r, such tumor c-115 have undargone ~lRni~icant chan~e~ ~ro~ th~ orl~inal pr~mary tumor cells, ln beco~ne atapt~d to propagA~lon i~ animal~. In addltion, it i5 ~ot t~ d 3Nd~a t~ 3~1Y~ EZ: 9T 06, Zla ~n~
. ..

.

~:~22~7~
~2-certQln or ~tr~ight forw~rd proc~ to ~n~rata ~uch tran~pl~ntablo tumor cell~ li.naa, ~in¢- only ~
~raction of prlm~ry tumor cell~ wlll have tho ability to b~ at~pt~d in thi~ wa~.
The dif~lcultio~ ~re ov~n gr~ator i~ ono attompt~ to cro~ 6pecle6 barrler~ in such tran~plQntablc tumo~ c~ . Human tumor c~ or example, will norma~ly sucoumb to the immun~ ~y~tem of mice. One w~y th~ has b~n d-v~lop-d ~o overcome thl-, in certain c~cumst~ncco ~or certaln human tumors, i~ u~e oP tho 'nude' mou~ ~train ~.P.Fl~nagan, Gon~t. Re~ mb. (1966), 8, 295-~09.
""Nud~," a n~w hairloss ~ene wl~h plQlotroplc ~ffect~
ln the mouoe."). In ~he~ mlco, d~ct~ in th-immun~ ayst-m ~llow thQ s~r~ival o~ cere~in forei~n ~ells i~J~ctet or tran~plan~ed into the anlmal.
~owover, ~ven ~r~ only a varl~blc fraction o~
primary human tumors can be ~uccos~fully propa~at~d.
Thu~, there i~ Q need for e~f~ctlv~ malntonanc~
and pxops,gation of primary human tumor c~ ln An ~nimal mod-l.
In accordance with an a~p-ct of t~ prea~nt invention, human c~ncor c~ are adminl~toret to, ~nd maintA~ned in, a non-hum~n anim~l wher~ln th~
non-hum4n ~n~mal i~ ~mmunod~fi~lent.
In ~ccordance with ~ prefe~r~t embotim4nt of ~ch aspeot o~ th~ pro~ent lnvcnt~on, t~a humsn cancer oell3 aro m41nt~ined in th~ non-hu~an anim~l wh~ch i- ~mmunodoflcient wlt~ ro~p-ct to itJ own lmmune ~yst-~, on a bioco~patiblo oupport which m~y inclt~ 4 growth Pacto~ or ~actors Por inducin~
An~lo~neui~ to the impl~nt, ~nd which may bo requlred for the ~rowth of the c~nc~ c~
(Blolo~ical R-~pon~ Mot~fior~, ur BR~

S ' d 3Nd~a ~333W0 bZ: 9~ 06, Z0 3n~

~22~70`

In aceordanc~ with another ~p~et o~ the p~ent inv~ntlon, human caneer coll~ a~ m~int4$~et in a non-h~man animal on ~ ~iocompatibl~ suppo~t whieh may lnelude such an~io~,enic or growth faato~ (BRM' The nen-human animal may or n~y not be an lmmunodefleient a~$mal.
In ~eeordanee wi~h a f~rthe~ a~poet o~ the px~s~nt in~entlon, th~ non-hum-n anim-l whioh inclu~o~ humAn canc~r cell8, in aeeord~ne~ with ~n ombodimont ~o hareln~bove da~cribe~ employet 8~
an animAl model ~or ~tudyin~ tho ~f~ot of a c~ne~r tre~tment or th~r~py.
In ~ooordanee with a further embodim~nt of th~
pre~ent lnvention, a non-hum~n animsl whieh ineludes human cancQr cellA in aeeordance with ~n ~mbodlment horoinsbov- d-ocrlb-d, ~urther lncludo~ a human immun~ ~y-t-m, in pQxtioular th~ anlmal i~ pro~ld~d with hum~n h~matopoi-tie e~ whleh ero~t~ ~ h~m~n immune ~yst2m in tho non-human ~nimal.
The no~-human ~nim~ pra~er~bly a rod~nt.
A~ h~r-insbo~Q indicated, the hum~n e~ne~r eell~
~4 pre~rably m~int~ln~d in a non-human anlmal on An implantod bloeompatlbl~ support whieh may in~lude BRM'~, a faetor or f~etors to lnduea ~nB~o8~ne~is ~nd eell growth. Thln implant 1~ somotim~g harelnafter re~rred to a~ ~ n~ov~ac~larlzatlon devlc~.
Th4 biocompatibl~ suppo~t may bo ~ blodeer~dabl-suppo~t ~om~mQs roforr~d to horoin ~ An ab~o~b~blo su~port) or nonblod~rat-~le ~pport (somotl~ ref~rr~d to h-r~ln ~ a non~b-orb~bl~
~upport), pr~orably a non-biodegr~dabl- ~upport.
The n~ovA80ularis~t~on d~v~c~ or ~4Vic-z ~n ~l~o include in add~tion t~ A blolo~lo~l ro~pon~
modlfl~r nn extraccllular m~trl~ componont or component~. Th~ ~upport may b4 form~d ~ro~ ~n 9'd 3N2~ 3iX19 ~Z:9~ 06, Z0 sn~
. .

`22~7~

oxtracellular proteln or t~o ~tr~-c~llular matrix component m~y be a~d~d to th~ ~upport. For c~smple the ~upport may be form~d Prom colla~n whlch ~8 4n ~xtracellular protein or an extr-c~llulax component may b~ addad to th~ ~upport.
A biolo~ic~l r~ponse modlfier tBR~ a biochemical asent th~t directly or ind$rRctly induco~
a chAn~e ln ~ene ~xpresaion et th~ cellul~r level.
Growth hormnne~ ~n~ th~lr chlm-rlc d~riv~tlv~ servo ~ cxumpl~ of a dlrect BRM; initiatln~ a blological respon~e ultim6toly dlrocted to the nucl~u9 ~nd mediat~d by a ~pecific hl4h affinity receptor.
Hydrol~q~ (plaRmino~en sctivators, colla~en~s~, h~p~r~na~ ervo as ~n ex~mple o~ ~n indlrcot BRM;
initl~tin~ ~ b~olo~lcal raspon~ by enzym~t~cally activ~tin~ or rel~a~ la~ont, ~tor~d or zymo~n prec~r~ors oP dlrect B~M~. The BRM may ~timulRt~ or ~acill~at- vR~culax ~rowth from a ti~u- os orga~, and may ~ required for growth of coll~ on ~h~
~pport.
Biolo41c41 r~pon~e modiflcrc u-sd on ~h~
~upport can b~ ~ns~o~-ni~ growth fectorc ~uch a~
HBGF~ BGF-II, pl~tolet-d~riv~d growth actor (PDGF), maoropha~2-dcr~v~d ~row~h ~ac~or (MDCF), epiderm21 ~ro~th factor (E~), fibrobla~t ~rowth ~actor ~OF), hypothalamu~-teri~d gro~th ~acto~
(HDaF~, rotlnA-t~rlvat ~rowth ~actor (RDOF), and mix~ure~ ther~o~. A pr~4rret ~mbcdim-nt o~ ~he lnventlon for inducine angio~o~esis u~e~ H~O~-I.
Deslr~bl~ hwtrolaso~ inclu~ a m~mber aoleQtot ~or tho group con~lYtin~ of heparln~e, colla~ena~, plasmin, ~ pla8mlnogen act~vator, thrombin, hep~rin~e, ~nd mixturQ~ thereof. Hermo~ uch the ~rowth f~ctor8 ~r~ pa~tlcul~rly d~ir~bl~ 4~

~ d 3N;~ 333W~ SZ :9~ el6, Z0 ~nY

2~2~

baologlc~l reopon~e modl~i~rs. Hormono~ p~cl~ically eliclt cell ~rowth and dlf~er~ntiation.
The neov~ocul~ri ~tion product c~n ~lco Include an extracellular m~trix componont, which may bo the materlal ~om whlch th~ aupport 1~ ~ormod or may be added to the ~upport. Althoueh appliaant do~ no~
int~nd to b~ bount by thcorotlcal rea~oning, it iD
bellevet that th¢ inc~ion o~ th- ~xtr~cell~lar matrlx component may, amon~ other ~unctlon~, functlon to hold and ~o prot~ct th~ BRM whioh 1~ in~lu~d in the product; i.~., prDvid~s for 8d~0rpti~n ~nd ~dheronce of the ~RM to the ~upport and poo~lbly p~ot6ct~ again~t dagrad~tion of the BRM. In at~lt~on, th~ oxtrac~ lar m~t~ix componont may unction to ab~orb cella which pa~tlclpat~ ~n the necv~scularization or t~mor c~ tded to the devlce. One o~ more ex~racollular m~trix compon~nt~, whieh may bç u~ed alono or ln combinationl include colla~en~ minin~, ~lb~onectin~, ~el~tin~, ~lycoo~mlno~lycans 1 glyooprotoin~ ~ prot-oglycAn~ J Bnt mixturo~ t~eo~. Tho m4st d~ir~ble xtrQ~ollular matrix compon-nt~ include gelat~ns ~nt colla~n~. A
proferred om~otimont uoe~ coll~gen Typa IV. Collagen Type IV i~ ~e~irable ~ocaus~ it provid-~ da~ir~ble ad~orption ch~r~ctorl~tic8 for th~ biolo~ioal ro~pon~o modiflor.
~ ho ~upport may bo eithcr ~n ~b~orbablo (biodegradable) ~upport or non-ab90rb~blo (non-biodograd~ upport and tho ~upport c~n b~
form~d from a extracsllular matri$ co~pon~nt. Th~
pr~f-rred ~upport i~ a no~ab~orb~blQ upport whlch includoo at l~a~t on~ oxtr~c~llular m~trl~ co~pon~nt to which a~ l-a~t ono BRM m~ be ~b~orbed. Examplc~
o~ ab~oxb~blQ ~upport~ includ~ memb~rs o~ th~ ~ro~p consi4tin~ o~ colla~n Type ~; known com~excl~lly ~y 3Nd,~a ~3~3W9 9Z:91 06, Z0 9 ~2~7~:

~. .
the trade namo "Gel~oam", l~minin, ibronectlns, ~elatin, ~lyoo~mino~lycan, ~l~collpldJ, proteolipid~, mucopoly~acohariteu and mi~tures thorooP. ~xampl~ of non~ orbable matricc~ includ~
member~ of tho eroup co~iatin2 of nyl~n, rayon, dacronl polypropyl~ne, poly~thylene, polyureth~nel exp~nded polyt~tr~luorethylene tPT~E~ cro~-llnked collaso Type IV, and mixturo~ th~-o~.
The 0upport 19 on- which i~ biocomp~ le with the hoat ln which it i~ ~o ~- lmpl~nted and h~ th~
rigidlty ~nd lltren~,th to ~upport ~ro~th of the add~d cell~.
Expandod polytetr~luorethylon~ (PTFE) haa been found to be ~ particul~rly ~uita~le non-ab~orb~bl~
~up~ort. Thl~ aupport provitc~ th~ followln~
bene~ita. PTF~ hs~ a ~ner~l laok of ~n infl~mm~tory r-spon~e whlch i~ th~ bA~ or tho current acceptance of PTFE in the ~rglcAl com~unlty. PTF~
can bo co~ted conveniently with ~xtracellul~r m~trlx componentB whiah can Ab~arb ~ blolo~c~l r~spon~c motifioz. Bloloeloally acti~o HB¢F~ ~nt HBGF-~ can be lmmobilizet to co~ en-coat-t PTFE by pr~vlou~ly e6tabli8h-t method~. PTFE polym-ro ar~ routln~ly en~inocr~d ~o ~arloua apeoificatlona to me4t ~ -multi~udo o~ requlr~d confi~uration~.
Non~woven multifilament an~el-hair ~ib~rs of expandod ~TF~ ar- comm-rclally ~vailAbl~ ~rom W. L.
~ore and Aa~ociate~ c., Flae~taff, ~rizon~ and ar~
p~rticularly suitabl~ B~ a ~upport. Th~-- flbor-allow ~uffici~t or~ani~dd ~roa fo~ infil~rati~g ~oll~ to be ~xpoaod to th~ ~nvironm-nt of tha ho~t.
Thl~ p~rmit~ tho fr~ ~xchan~- oP nutr~ntd ant to~ic wa~to to oocur ~hllo noov~c~larizAtion prooo~a occur.

6'd 3Ni~E[ t:l~l3~ 0 9Z:9~ 06, Z0 sn~

~2~70 . .

8upport~ can be provided for u~e in thio inv~ntion in ~ny da~ir~d Ehapo and alze. I~lrable ~hap~ for a ~uppo~t c~n b~ a ~trlp, ~ ~pcnge, or a ~ube. Supports sr- c~o~irai)ly cap4bl~ of boin~
~c~d wlthin an orl~;aniam. ~ult~l~ moano for oecurin4 ~ ~upport can includ~ a ~ple, biocompatlble glue, or other ~rgic~1 proc~duro~ auch a~ suturing or tyin~ thfi ~upport to a tia~uo.
A de~r~bl- support i~ obtaine~ by ~ilin~ a tub~
or ~ ve of ~xp~nded PTFE with ~xpandcd P~Fl~ ~lber~
or "an~1 h~ir"

For ~xample I HBaF~ known to bind to component~ oP the ~xtraco~ r m~trix an~ component~
o~ the æxtracelll~lar matrlx can elth~r ~- lmmobll~æed on ~olid ~upport~ or donatur-d ~nd fo~m~d into an in~olubl~ mat~ix ~BOF~ d~orbed to ith~r denatured coll~en I ~pong~, or to flb~ro o~
expQnded PT~ co~ted with ol~har fi~ron~ot~n, coll~en I or colla~an ~1 or to combin~tion~ o~
d~n~ture~ sponge~ wr~ppQd wlth PTF~ ro, promoto0 6ngio~no~i8 ln both tho normal and lmmun6 ¢ompromi~od (BN~) mou~e ~t cone-ntr4tion- which ~r~
conoi~tont w~th the growth, ~ b~low ~ct4r'9 Ae~iviey ~ a cellul~r ~ito~en in vltrQ
Th~ most ef~ecti~4 conc~n~ratlon~ for ~
biolOglCAl re~pon8c modlfi-r~ eAn be 8 ~ono~ntx~tion that ~licits ~ growth responsQ from tho t~r~
but i~ not toxle to tho~a e~ Ef~aotlvQ or th~rapeu~ic ooncentration~ o~ anSiogonio ærowth factor3 ~r- betwoen about 1 to lt)O nanogr~8 p~r cublc mil~imct-r o~ a ~upport. ~ ~upport for thlll calculation i~clude~ both ~h- ab~orbl4ble ~uppo~ ~nd ~he non-ab~orb~lo ~uppost.

01'd 3N~ 1132~ 8Z:9~ 06, Z0 9 .

" 2~22~7~

In ~ocord~nce with an a6p~c~ of the pro~e~t inventlon, 4 devio~ o~ the ~ype her4inebo~0 d-~crlb-d 19 ~eeded with hum4n c~ncQr cOEllo prior o~ ~ub~eguent to impl~n~atlon of the d~ico in ~ non-human anlmal.
Th- non-human anim~l~ wbich aro providod wi~h ~h~ human cancer cell~ are preferably i~munod~flcl~nt non~hu~an animalo; in particular immunodefici~nt mice and ra~s. ~uch immunode~lci~nt mic~ ~r~ known ln the art snd may for ~x4mplo b~ SCID mic~ or mice of the beigeJnulXid, ~BNX) e-notyp~, ~McCun- ~.M., Namlkawa R., Kane~hima H., Shultz L. D., Llberman M., Woi~m~n IL: Tho "SCI~Hu Mouse: Murinc Model Yor the An~ly~i~ of Hum~n H~matolymphoid Differ~ntla~lon and Funct~on" Scienc~ 16S~-1639 (1988). Mo~i~r D.E., Gull~ia R.J., Baird B.M., Wil~on D~
"Tran~f~r of ~ Pu~ctlonal Immune By~tem to mic~ with S~v-rOE Combined ImmunodQficienay". Naturo 33S:
256-Z59 (1988). Kam~l-Reid, S. and Dlok~ J.E.:
Engr~ftm~nt of Immune-Deficient mice with H~man Hematopoi-tic otem cell~, Scien¢e 242: 1708-1709 (1988)~ and which h~vo little i~m~ne function left to interfer~ w~th the ~rowth of forelgn c~ . The~e mico h~ beon oxten~iv-ly u~-d for human tumor X~no~rA~ Isai~h J. Fidler1 C~ncor and MQta~ta~a Re~lew, 5:2g~49 (1986~, "Rational~ an~ mothod~ for tho u~ of nuto mice to ~tudy ~h~ biolo~y and therapy of human c~nc~ m~ta~ta~iD"; 5~1~i Nai~o, ~
Clin. Expl. M~ta~A~ls, vol. 5, ~2, 13S-146, (1987), "Growth and mcta~tatic b~havior o~ hum~n tumor ~ells implant~d into nutQ and bel~o ~ud~ mlo~,"; Jur~en Matt~n~ Q~ Canaer ant M~ta~t~8 Rovlew~, 7:263-284, (1988), "Hum~n tumor x4nogra~t~ a~ mod~l o~ drue teatln~ ntr~w P-wlowa~i Ant Pot~r J.
LQa, 1n S~in Tumoro: Exporiment~l ant Cllnical Aop~ct~, edited by Claudio J. Conti, et ~ , R~ven I~'d 3N~:U~a till3w~ 8Z:9~ 06, Z0 9n~

~22~7~

Pra~, Uew Y~rk, (1939)~ "Hum~n M~lanoma X~no~rafts".3.
Al~hou~h in a preferrod l3~bodlmcnt, th~ hum~n csncor c~ are plQo~d on a neovaocularis~tlon ~evio~ o~ tho typ~ d~scribed in ~n lmmu~odeflcisnt non-human ~nlmal, it i~ posoible within the 9pl~it and scops of the invention to use a non-immunodeflcl~nt non-hum~n anlmal.
~ n accord~co with another ~mbodiment of th~
inventlon, the im~una dQficient non~hum~n animsl, in p~rticul~ m~co, ara provlded w$th other humsn c6~1 ln addltlon to the prlmary human canc~r c~
Fir~t, ~h~oa may be pr~cur~o~ cell9 th~t will provid~
the ~nim~l with ~ ~umQn immune ~y~tam. This klnd o~
dov~oo ~llow~ t~ting of ~ell~lar and ~$ologlcal proc~d~roo for arreotlng tumor cæll ~rowth or Por ~umo~ do~truction. In par~c~lar, exa~pl-~ arc adopt~v~ Imm~notherapy procedure9 8uch a~ LAK ther~py (Rosonbor~,&.A., J.N.C.I. 75, 595-603, 1985; Mule, J.J., et ~1!. Sci~nce ~æ~, 1487-9, 1984) or T~ coll thor~py (Ro~enberg, S.A,, ot Ql., New En~l,J,Met, 319, 1676-aO; Rooenb~rg~ S,A., et al., Sci~nae ~23, 1318-Zl, 1986.), or ~ny a~ent~ c~p~lo, o~
adm~nistr6tion, o~ ot~m~latin~ tho n~wly ~n~r~ted immune ~yotQm in th- mou~e to ~r~t th~ g~ow~h of, or ~ill th~ tumor aello in, th~ implant. ~hiD ~yAtem i~ ~ced in an autologou~ fa~hion e.~., th- immun~
~ystem of a patlent i~ duplicatet ln ~h~ ~mmune d~ici~nt mlce, w~ilo tumor c~ 8~ impl~nted ViA
the implfi~t, and then the pa~ticular the~apie~
appliod.
Second, other colls in ~dtltion to the tu~o~
cells m~y b~ ~pplisd in the lmpl~nt. The~ coult be any of ~ wld~ ~arl~ty of cell~ ~nown t~ ~e able to ~roW on s~cb ~ lmplant sy~tem, an~ may in~ludo, but Z~'d 3N~ a ~3i~ ) 6Z:91 06, Z0 9n~

~Q~7~

no~ be li~ite~ to, ibroblaste, smooth mu~clo cell~, endothel~l coll~, eplthollal cell~, h~p~tocyt~, me~othQli~l cell~. Such cell- m~y funotion to ~pport tumor cell growth by 1~ v~rioty o~ m~chanl~ms eg by ~oaretlon of particul~r ~rowth ~actor~, an~io~en~c f~ctor~, extracollul~r matrix ~ompon~nt8, or other ~ctor~, all o which m8y con3titut~ the approprl~e micro onvlronmone for ~int~n~nco of the properti~ and ~row~h of the pr~mary tumor cell~.
A4clitlon~ , cell~ may be add~t and ~cr-i~net ~or the propsrty o~ production of a~nt~, or oth~r prope~tiao of lnhibltlon o~ the ~rowth o~ tumor cell~ which usuall~ erow well on the support without add$tional c~
The human canc~r e~ whlch ~ro u8ed in the pres~nt $nvention arq oo-callod primary human canc~r cells (i.e.l oell~ t~en ~irectly from ~ patient'~
tumor ~nd maintain~d i~ vltro only ~or th~ ~horte~t po~iblo time). The c~ncer c-llJ may lnclud~, but are not limlted to th~ ~ollowln~ typea; brea4S, lun~, ovarlan, melanom~, colorect~l, ronal.
Thu~, in acco~dancQ with Qn a~p-c~ of t~
pre~n~ in~ontion, hum~n c~ncor collJ ~r~ plac~d in B
non-human nimal in an la v~vo phy~iolo~lc~l ~nvlronmont th~t m~intain~ th~ Approprl~to propo~ti-s and &has~cteri~t~c~ of thæ ori~inal human primasy canccr cHll~. ~n thi~ mann-r, ~uch human cana-r cells may be t~stad and/or ~t~di-t ~n~/or troat~d in ~ non-human anlmal ~odHl und~s $~ Y~XQ condltlon~
w~l~h do not 61ter tho ch~ract~ tic~ of th~ c~nc cell~.
Thu~, the ps~-nt invention c~n provlt~ ~
non-hu~an ~n~mal model ~y~tem for t~stin~, ~tudyin~, m~nlpulatin~ And prot~cting behaYlor of tumor 1'd 3N~ tl333W9 6Z;9~ 06, Z0 9nt . . .

2~22g7~

and devnlopine tr~atments for prlmary human tumor call llne~ ~rom a vari~ty o~ dlfforent cancers.
In accortanc~ with ~ho prQ~ent inYentlon, the propertl~ oP cell~ o~ ~n in~:~vldual human patl~nt may ba dovelopod in the anima:L modol and be u~ed to pr~dict the gr4wth ~nd b~havior o~ ~uch cell~ in t~e pat~e~.
In addition, R patlnnt'~ tumor c~119 m~y be Qs~blish~t ln n nu~bcr of dlfP~ront non-humQn 4nimals ~nd ~uch anim~l~ may bo ~ubJ~ctod to ~
vRrioty of thsrap~uti~ procodur~ ln~ly or in combinAtlon; chemlcal ~nd~o~ phy~ic~l) to thR~oby d~t~mino th- bo~t cour~ of tr~atmont.
~ ho invention will bo further d-~rib~d w~th respoct to th~ followin~ ~xamples; however, the scope o the lnvont~on i8 not to b~ limited ~h~r~by;
~x4 pi- I
Thc abdom~n o~ an ane~thct~zod mou~e wa~ wa~bet wlSh 20 perc~nt etha~ol ~nd an inci~lon w~ ~ade into ~he abdomincl wall to oxpo~e th~ peri~onaal c~ity.
:Cel~oam (UpJohn, Kalam~zoo, Michi~4n), cut into ~trip~ of appxoxim~t~ly 20x15x7mm, or expant-t PTPE
fiber~ (W. L. Cor-, Inc., Fla~tQff, A~i80n~) pre-coatet with colla~en IV and prc-wei~h-t t0.2~m dry w-l~ht). or ~ combina~ion of the~o two mato (Gel~oam ~trlps wrapp~d with PT~E f~ber-) wore t~ted with approximately 0.5 ue o~ ~BGF-I in 100 ml of w-t0r at 4DC ~o~ one hour. Pollowin~ ad~orptlon of the ~rowth ~oto~ the ~on~truct~d l~plant devico wa~ placed ~d~acent to ~he llYer o~ th~ mou~-. Th~
~u~loal openin~ W~9 clo~ed by ~uture or stspls eu~
and tho mice w~ro ~aint~lned on ~ norm~l ti~t Rnd 12 hour li~htldark cycla. At v~rlou9 tlm~ po~t implant (18,Z8,3S and 38 ddy~) the mlc~ wero klllod ~nd then ~I'd 3N21,~EI tl333W3 0E:91 0~, Z0 9n~

2~22~

~llb~oted to wholql bo~y pr~ ur~ pcr~u~ion with glutaraldehyde, b~f~red with p~oYphato, ~sin~ a e~theter in~rted into the thoracic aor~a. The lmplant~ wero exfiminod ma~roscoplcally for blood ve~l formation ~nd the int~rn~l or~an block remov~d ant thin ~ction~ throu~h the en~lr~ organ block contslnin~ the lmplant devic- w~rc pr~par-d ~or hl~ologlo~l QxAmination.
A ~gnlflcsnt anglog~nic re~pon~e w~s oh~erv~d macro~copically and mlcroscoplcally withi~ 18 days ~fter su~ge~y in all mic~ tnorm~l ~C57) ~nt imm~no compromi~d (BNX) mlcQ) treated wlth HBOFI. ~lood ve88~18, ~t the ti~su~ ~lte of impl~ntation, wsre obs~r~d macro~coplc~lly to b- ~xclu~ivoly within th~
~mplant. Control impl-nt~ without HBGF-I tid not induc~ neovascul~riz~tion. Hi~tolo~ical 4x~m~nation of ~ho implant5 r~v~led new blood ve~el ~rowth withln tho implanta. Iotal numb-r~ of ~ic~ ~n~lyz~d in thi~ ~p~riment w-r~ 3 for C57, and 18 ~os B~X.

After ~ lon~ a- ~0 day~, th~ lmplant~ wor~
ex~mined for th~ extent o~ ~ngio20ne~i8.
BidirectlonAl formatlon of n-w blood v0~8018 ~lone thc HBaF-I lmpl~nt fro~ tho l~vor and o~hor ti~uc ~ite~ w-re ob~rvet. Further xamln~tion r-v~led that thc implant containod v4~c~1ar "~trln~" which conn~ct~d thc implant to the oxi~tin~ va~cular tr~.
Tho abi~ity o~ HBCF-I impl~nt~ to m~lntain th-~neovo~-l ot~uctur~ wl~hin the p-riton-um 1 evidancod by these h~hly va~cular brld~
Hi~tologioal ex~mlnAtion of theo~ lon~ t~rm impl~nes di~play~d ~ lar~o or~anlzed ~ol$d matsix urro~nded by a colla~enou9 c~p~ulo (~car) Ant containing a network o~ noove-sel ~orm~tion~ inte~dle~tated with dlf~rdnt cell typeo. Hl5tolo4ical oxamlnat~ono ~-d 3N~U~E[ t;l333W9 ~:91 06. Z0 911b~

... .

~Q2~7~

(lon~itudfnal ~nd cro4~-B~c~on) throu8h typlcal ~scular "~rln~" reve~lod th- pres~nce of ~
monolsyer n~ me~otheli~l cell~ qurroundin~ a lar~o vascular lumina in th~ central port~on, encompa~ed by prominont endoth~llal celln and multlplo l~yer~ of : ~mooth mu9cle c~118~ reprc~ontlng maturs 4nd hl~hly differentiat6d art~riol~s. Withln Sho perlphory ara ~bundant caplllary lumina, snd tho ontlr~ v~aular bundl~ i~ fiurro~nd~d by B continuou~ ~bro~ellul~r cap~ule. P~ther examlnation o~ thl~ ~ample ~t hl~h maRnific~tion r~v~alet th~ re~atlv~ly ~ich coll~n component o~ Y~scul~r ~tructur~ a~ w~ tho abundAn~ of ~ndothall61 cell-lin~ o~p~llsr~
~tructur--.

In ~ccordanc~ with th~ d~vice ~nd mo~hod of th~
preoent invcnt~o~, ~n~log-n-~lJ ~nd n-ov~cularis-tion h~ b-en ~hi-v-d in th~
peritoneal o~Yl~y o~ the lmmuno- compro~od mo~o ~BNX).
~a~pl- 2 Examp1e 2 demonstr~tes that tho pre--nco of a 1~r~- v~cul~riz~d, or~nizet ~olld matrlx, which contaln~ a network of neoves~l formatlona, contiguouo wlth tho ~NX mou~e va~cl~ tra~, pormit~
auccoJ~ful tran~plantation o~ oith~ a r~t-t~ri~d hQp~tom~ coll lin- or ~ human-dorivad molanom~ oell lln~ into a ~NX mou~e.

Th- diploid co11 strain, RL-PR-C, ~ mi~lmally de~iatet rat h~patoma ~oll lin-, ~nd 8 coll line, ~375, d~riv~d from a human melanom~. ~er- m~int~in~t ln culture ~ith ~lth~r RPMI 1640 (RL-~RC) or hlgh 91 d :~lNii~ tl333~1tl3 ZE :91 06, Z0 3nt~

- s~ ~ s~ , Q

-1~
Rluco~e d~in~d ~nlnimnl ~ nt:i~l medla wlth loZ
~et~Ll cal~ ser~m, 0.~3M E~lutamine ~nd 8ent~miain (O,S
mS~lml). Th~ c~ w~r~ pl~t:~i on lOOmm tl~ue cl~lt~re plat~s an~ maintained in c~lturQ with m~dium changed every two (2~ day~ for th~ ~u~tlon o~ th~
e~:perlm~nt .

In ordor to id~ntl~y Imd ch~ra~terl~ tho tun~or cell~ ~uring th~ ~ran~plnntation proc~, c~
wGro t~n~d-lc~d with recombin~Lnt r2~roviral vector~.
~ither N2 or LNL6 (Armeninano , ~ ., et 1l1 ., J .
Ylrolo~y 61, 1657-1650; Adam, ~.M., ~nd Millar, A.D.
(19~) J. Virol. 6~, 3802-3B06) wa~ u~d to tro.n~~r d lnteg~te the ~enq for n~omycln r~slstanc~ (NooO) into th~ ho~'c gonomic ~quonc~. Both o~ thQ
ret~ovlrAl v-ctor~ ed in thia ~xamplo arG Moloney murin~ leuJF.orni~ viru~-b~flod v~otor1 wltb the ~ al codlne sequenoes removod and th~ Nao-ro~i~tanc~ ~ne ~rom th~ ~acterial TnS transpo~on ln~rt~d. A clonQ
produoin~ a hleh tlt~r of amphotropic viruo containin~ h~x N2 or LNL6 wa- loolat~d by C418 8electlon of the helper-fr~e p~ck~ing coll line PA317 a~t~r ~lci~m pho9phate-motiated DNA
'cre,no~tion o~ the appropri~te vlr~l pl~mids.
Vir~-oontain~ng ~up~rnatant~ wcr~ col~ect~d from a con~l~en~ monolayer in Dulb-cco'~ minlm~l o~Henti~l m~dl~m cont~ining lOZ fetal oal~ ~orum. Followin~
fi~trationl the viral supern~tAnt~ wer6 ~torcd A~
-70~C un~11 u~ad.

Culturat monol~yars o~ ~lth~r PL-PR-~ c~ or A375 c~ (2~1Q6~ Wa~ po~d to vlru9 cont~inin~
suporn~t~nt~ ~on~Aining v~ru~ part~cle~ (2x106), polybrene ~8ue/m~) and appropriAt~ cultur~ m~diu~ for 2 hour~ at 37~C. Followi~g expoour~, freMh a~lture ~-d 3~a ~3w~ z~:s~ 06, z0 9n~

7 ~

m~dium wao added and cells w~ culSur~d . or 48 hour5 ~t 37C~ In~ected cellæ, ~oth ~ -P~-C ~NeoO) and A375 (Neo0) wer~ oxpo~od to G41~ (800~/ml) for two week~ wh~rouposl tre,naduc~d eolonio~ wor~ ~xpanded ~or ~ubs~quent tran~pl~ntfLtlon.

TrAnJclucet tumor c~118, R~-PR-C (Neo~) er A375 (N~olD) w~rQ ~rown to hl~h d~n4ity (lx10~) on G~l~oJ,m sponl3e~ ( 15x12x7nun) and wrapp~t with O . Z~m of expanded PTFR ~ibcr~ prcviou31y co6ted wiSh coll~ n IV and containin8 Ad~orbe~ ~IBaF-I ~0.5u~). Thi~
device wa~ ~urelc411~r implanted in the perlton~Al ~avity ~d~ac~nt 'co the liver oP ~NX mico a~
p~ riou81y d~ocrib~d.

~ t v~rio-~ tim~8 po~t impl~ntation, wrapped ~pon~e~ Were a~r~icAlly romovQt and wa~h~d ex~n3iv~1y in pho~phat~ bu~r~d ~lina (P~S~.
Ws,~h~d implQnt~ w~ ostec~ 30 to 60 toinu'c~s ~t ~C Wlth a ~o~ ion o~ colla5~-n--e in 1~ (lmg/ml) in a ti83uo cultur~ incubator (5% C02). Rele~sed c~ wera collecte~ by centr~$~l~aSlon (~0 min, 2000 x ~, 20~) an~ w~0h~t onco with PB~ ~nd p~llot~d ~y centrlfueation. Cel~ woro roouspend~d With appropriate ti~au~ cultur~ modium cont~lnin~ G418 ~800ug/ml) and platæt fo~ 16 ho~r~ on lOOmm tissue oulture ~lfl~k~. Plated ce~l~ Wer~ wa~h~d with PBS
exten~iv~ly ~n~ ~d P~-0h metium. Madi~ w~6 chan~sd e~cry tWo day~ ~or th~ dur~t~on of thc proe~dur~.
L~e number~ of ~iablc ~L-PR-C (Neo~) call~ wor~
~ecove~ed fl~ 18, 2S and 34 t y8 po~t i~plant~tion a~
~vidonced by th~ir abili~y to ~row in th- pro~nc~ of B418. Th~3 oboorvation wz~ ~urthor coni~ od by cL
tirect asssy o~ th~ n~omycin pho~photra~
activity (th- d~ rect p~od~ct of th~ NeoO ~no) .

8~'d 3N~ 3~ EE:91 06, z0 ~n~

~h~70 ~Tiable A375 (N~o~) ooll~ wer~ r~cov~red 8t 27, 34 ~nd 40 tays po~t lmpl~ntation which both grew ln the presenc~ of 04~8 a8 w~ll as di.~plsyed pollitiv~
neomycin phosphotrans~ers,ae aet~vity, Indu~d noova~cularizaticn within ~h~ poritoneal cevity ha~ therefor~ boen shown ~o ~uataln th~
proli~erative pot~ntial of tumor cell~ simult~neou~ly imp~anted with ~IB~;IF-l 6d~0rbed ~olid ~upportu. Turaor celln which were recov~red from thcoe HB~;IF-I implants w~ro able to proliferate ~ yitro und~r ~oleotlve pr~fiour~ which refl~ct~ thoir ~2n~tlc dl~posltlon.

Prim~r~r tumor ~mples ar4 obtdin~d a~l biop~y s~mpl~ from hu1nan patients.
rh~sa tumor call~, purl~iod from inmuao ~yst~m cell~ if n~c~ex~, by ~t~ndard coll--aparatlon proc~d~ros or as pioc~- o~ in~sct tumor, ar~
main~ined for ~ minimal p~riod ~ ~Q in arl appropriatQ m~tium prior to ~din~ onto th- ~upport for an implan'c, ~o del~cribod in Exan~pl-s 1 and 2.
The oell~ on the s~lpport aro imp1antod into BNX mlc~
aa previously d~llcribe~ or aa O . 2S eo O . 5 aub~c centlmet~r chun~e~ wr~pp~d in O. l ~n ~iber~ coated wlth collag~n I ant O . 25 ~Ig HBCF~ nd ~ tim~ cour~e of ~llsvi~o.l of th~ c~ n tho lmpl~nt 1~ ~a~rlocl o~t. Th~ lmpl6nt io plac~d in the poritona~l c~vity.
Sur~lv~l ~nd growth of th~ c~lle i~ nlonltore~
bioch~mic~lly, by i301~tion DNA ~rom c~ in th~
implant, and moleoular hybrldl~tion w~th human-~pecific repetltiv~ ~equenco DNA prob~.
Monitorlng i~ al~o by immunohi~toch~mlc~l mo~nn, l~ing lnopl~nt ~octiono ~nd l~ranune antlo~ra apecific 6~'d 3N~ a ~33~ 10 l7:91 06, Z0 9nt~
. _ 7 ~

for human and/or e~lmor cell ~p~clfic gurfa4~ marker~.
~lnally, mou~ rum can be ~cre~nat ~sr ~ny human ~nd/or tumor ~peci~ic ~ec~tion p~oduct~ made by c~ on ths lmplant and s~crated into the clrculation .
Havin~ 04tsbli~hed primary human t~mor c~ on impl~nt~ in a ~erles of BNX mic~, various therapeu~ic modallties can b~ t~st~d to ~co which arQ moEt e$~ectiv~ in killin~ of the patiant' 8 tumor cell~ ~a ~iY~ ln ~he moua~. Th~ trcaem~ntc c~n b~ but ~r~ not llmited to resul~r ch~moth~rapy with ect~bll~hed a~ents, radi~tion th~rapy, treatment wlth blologic~l respon~e modifiera and an~itumor ~nt~ 0uch as tu~or necro~l~ ~ctor~ int~rl~ukin 2, ~ lnto~ron, inter~ron, interl~ukin 1, interl~ukin 4, or a wid~ ran8~ of oth-r ~ch a~ant~.
Re~pon~a to ~h~rapy can b~ monitorQd ln saYoxal w~a. Mo~t oim~ nd efrecti~e 1~ to ~onitor a docrea~e ln productlon and ~ppearanco in the mo~e ' P
clrculatio~ o~ tumor ~poci~lc product~. Also, th~
implant~ can be ex~min~d biochomi&~lly ant hi~tolo6ically to dotermln- the ~ur~ f th~ tumor col~ .
~ dditlon~lly~ hum~n bono marrow can b~
introduced ~nto tho BNX mice by publish~d procedure~
(~e~ ref*~ncc- ~arll~, AO ~ bono marrow tr~n~plan~, to ~#t6bll~h a paticnt'~ bon- mRrrow in th~ ~mmune d~ici~nt mouo~. Th~n t~or c~llo can bo lmplant~dj ~9 do~crib~d ~bovH, and a varl~ty o~
~dd~tional canQ~r thor~pi~s, in~ludin~ ~h- s~op~
lmmun~th~rApy ~pproach, ~an bæ te9t~t. Tho~- ln~lud~
L~K cell thsr~py, TIL eall thorapy and ALT ther~py.
urvival of tu~or cell~ 1~ monieorod ~8 d~sc~ib~d ab~v~. Th~ adoptiv~ immunother~py proc~dur~ o~ LAX, 0z d 3N~a ~3~ 91 06, z0 sn~

TIL or ALT c~n be tost~d with ~h~s patlentd immun~
C8~ llg alane, or in ~ mouse wher* th~ patierlt ' ~ own bone marxow has ~oen r~con~t~tutQt $nto th~ mou~e.

Numb~r modifica~ciorLs and vsriQtion~ of thl~
inven~iotl ~r0 po~ible; th~r~:Eor~, Withl~ tha ~cope o~ tha dopendent clain~, th~ lnventlon m~r be practiced othorwise than a~ particularly tlescribed, 3~ T13~ s~:s~ 06, zz ~nt~

Claims (32)

1. A process for producing an animal model containing human cancer cells comprising:
providing a non-human animal with a biocompatable support which includes both biological response modifier and human cancer cells.

2. The process of claim 1 wherein the non-human animal is immunodeficient with respect to its own immune system.

3. The process of claim 2 wherein the biological response modifier is an angiogenic growth factor.

4. The process of claim 3 wherein the growth factor is a heparin binding growth factor.

5. The process of claim 2 wherein the support includes an extracellular matrix component.

6. The process of claim 5 wherein the extracellular matrix component is a collagen.

7. The process of claim 2 wherein the support is a non-absorbable support.

8. The process of claim 7 wherein the support is comprised of polytetrafluoroethylene.

9. The process of claim 2 wherein the non-human animal is a beige/nu/Xid mouse.

10. The process of claim 2 wherein the cancer cells are primary tumor cells.

11. The process of claim 10 wherein the biological response modifier is an angiogenic growth factor and the support further includes an extracellular matrix component.

12. The process of claim 2 wherein the non-human animal includes a human immune system.

13. The process of claim 12 wherein the biological response modifier is an angiogenic growth factor.

14. The process of claim 12 wherein the support includes an extraceullar matrix component.

15. The process of claim 12 wherein the support is a nonabsorbable support.

16. The process of claim 12 wherein the non-human animal is a beige/nu/Xid mouse.

17. The process of claim 12 wherein the cancer cells are primary tumor cells.

18. The process of claim 12 wherein the biological response modifier is an angiogenic growth factor and the support further includes an extracellular matrix component.

19. A process for testing a cancer treatment for human cancer cells, comprising: subjecting a non-human animal to a treatment for human cancer cells, said non-human animal including cancer cells, said non-human animal being immunodeficient.

20. A process for testing a cancer treatment for human cancer cells, comprising: subjecting a non-human animal to a treatment for human cancer cells, said non-human animal including a biological response modifier and human cancer cells supported on a biocompatable support.

21. The process of claim 20 wherein the non-human animal is immunodeficient with respect to its own immune system.

22. The process of claim 21 wherein the biological response modifier is an angiogenic growth factor.

23. The process of claim 22 wherein the growth factor is a heparin binding growth factor.

24. The process of claim 21 wherein the support includes an extracellular matrix component.

25. The process of claim 21 wherein the support is a nonabsorbable support.

26. The process of claim 21 wherein the non-human animal is a beige/nu/Xid mouse.

27. The process of claim 21 wherein the cancer cells are primary tumor cells.

28. The process of claim 27 wherein biological response modifier is an angiogenic growth factor and the support further includes an extracellular matrix component.

29. A non-human animal model produced by the process of claim 1.

30. The process of claim 2 wherein the support including the cancer cells is implanted in the non-human animal.

31. The process of claim 2 wherein the cancer cells are added to a support implanted in the non-human animal.

32. The process of claim 20 wherein the non-human animal includes a human immune system.