Immunotherapy of human tumour xenografts overexpressing the EGF

Transcription

1 Br. J. Cner (1993), 67, '." Mmilln Press Ltd., 1993 Br. J. Cner (1993), 67, Mmilln Press Ltd., mmunotherpy of humn tumour xenogrfts overexpressing the GF reeptor with rt ntiodies tht lok growth ftor-reeptor intertion H. Modjthedi, S. les, G. Box, J. Styles & C. Den Setion of mmunology, nstitute of Cner Reserh, Sutton, Surrey, UK. Sunmmry Athymi mie ering xenogrfts of humn tumours tht overexpress the reeptor (GFR) for GF nd TGF hve een used to evlute the therpeuti potentil of three new rt monolonl ntiodies (mas) direted ginst two distint epitopes on the extrellulr domin of the humn GFR. The ntiodies, CR16 (gg2), CR62 (gg2) nd CR64 (ggl), hve een shown (Modjthedi et l., 1993) to e potent inhiitors of the growth in vitro of numer of humn squmous ell rinoms euse they lok reeptor-lignd intertion. When given i.p. t 2 fig dose, the three ntiodies were found to indue omplete regression of xenogrfts of the HN5 tumour if tretment with ntiody ommened t the time of tumour implnttion (totl doses: CR16, 3. mg; CR62, 1.2 mg; CR64, 2.2 mg). More importntly when tretment ws delyed until the tumours were estlished (men dim..5 m) oth CR16 nd CR62 indued omplete or lmost omplete regression of the tumours. Furthermore, tretment with totl dose of only.44 mg of CR62 ws found to indue omplete remission of xenogrfts of the rest rinom MDA-MB 468, ut CR16 ws less effetive t this dose of ntiody nd only 4/8 tumours regressed ompletely. CR16 nd CR62 were poor inhiitors of the growth in vitro of the vulvl rinom A431, ut oth indued sustntil dely in the growth of xenogrfts of this tumour nd 4/8 tumours regressed ompletely in the mie treted with CR62 (totl dose 2.2 mg). Although CR16 nd CR64 were more effetive thn CR62 s growth inhiitors in vitro, CR62 ws found to e sustntilly etter t induing regression of the tumour xenogrfts due perhps to dditionl tivtion of host immune effetor funtions y the gg2 ntiody. We onlude tht these ntiodies my e useful therpeuti gents tht n e used lone without onjugtion to other ytotoxi moieties. The reeptor for epiderml growth ftor (GFR) is 17 kd trnsmemrne glyoprotein tht hs een found to e overexpressed in mny types of humn ner nd to e of prognosti signifine in ertin types of humn mlignny (Hrris, 199; Gullik, 1991). Furthermore, the finding tht mlignnt ells expressing high numers of the GFR redily form tumours in thymi mie points to orreltion etween trnsforming potentil nd the numer of GFR expressed (Snton et l., 1986; Velu, 199). videne tht the reeptor is involved in n utorine loop tht ontrols growth of these tumours omes lso from the finding tht mny primry tumours o-express oth the reeptor nd either of the lignds TGF or GF (Sporn & Todro, 198; Sporn & Roerts, 1985; Derynk et l., 1987; Di Mro et l., 1989; 199; Yoshid et l., 199; Kurhi et l., 1991). n ner ptients, the overexpressed reeptor my onstitute suitle trget on tumours for ntiody direted therpy (Mendelsohn, 1989; Hrris, 199; nnis et l., 1991). ndeed, in ertin rin tumours sustntil hnges in the externl domin of the GFR hve een found s onsequene of deletions in the genes oding for these regions nd the novel juntionl sequenes formed ould provide tumour speifi trgets (Stek et l., 1988). Antiodies direted ginst growth ftor reeptors suh s the GFR n t in more thn one wy. Firstly they my e le to inhiit growth y loking growth ftor-reeptor intertion nd seondly they my e le to reruit the immune effetor rm of the host to ring out tumour ell destrution. The ltter funtion whih inludes tivtion of omplement nd intertion with F reeptors on effetor ells is ritilly dependent on ntiody isotype. Over the lst dede, numer of monolonl ntiodies (mas) hve een rised in mie ginst the externl domin of the reeptor on the humn vulvl rinom A431 (e.g. Shreier et l., 1981; Wterfield et l., 1982; Sto et l., 1983; Fendly et l., 199). Some of the mouse mas hve een shown to inhiit the growth of humn tumour ells when ultured in vitro or when grown s xenogrfts in thymi mie (Msui et l., 1984; Rodek et l., 1987; Aoud-Pirek et l., 1988; Pellegrini et l., 1991). Also, linil trils hve een undertken using some of these ntiodies to the GFR (GFR-1, 225 or 425); rdioimging studies in ptients with hed nd nek ner (Soo et l., 1987) nd squmous ell lung ner (Divgi et l., 1991), rdioimging/rdioimmunotherpy trils in ptients with rin glioms (Klofonos et l., 1989) or mlignnt stroytoms (Brdy et l., 1991). The results of these linil trisl hve een promising nd point to role for some ntiodies in the detetion nd tretment of ertin mlignnies. Using two other well hrterised humn rinoms tht overexpress the reeptor for GF nmely, the hed nd nek rinom LCR-LON-HN5 (sty et l., 1981; Cowley et l., 1986) or the rest rinom MDA-MB 468 (Filmus et l., 1985), we hve generted series of rt mas ginst four epitopes (A-D) on the externl domin of the reeptor. The properties of these ntiodies nd their effets on the growth of GFR-expressing tumour ell lines in vitro hve een desried in the ompnying pper (Modjthedi et l.) or reported elsewhere (Modjthedi et l., 1992). The ntiodies tht ound to epitopes B, C nd D were found to lok the inding of the lignds GF nd TGF nd some of these (ginst epitopes C nd D) were potent inhiitors of the growth in vitro of rinom ell lines tht overexpressed the reeptor for GF. We report here the results of experiments tht show () these ntiodies lso prevent the growth in thymi mie of xenogrfts of humn tumours tht overexpress the reeptor for GF nd () the isotypes of the ntiody influenes the effetiveness of the tretment in vivo. Correspondene: C.J. Den, nstitute of Cner Reserh, Hddow Lortories, 15 Cotswold Rod, Belmont, Sutton, Surrey SM2 5NG, UK. Reeived 19 June 1992; nd in revised form 9 Septemer Mterils nd methods Cell lines The following rinom ell lines whih express the GFR were otined from Dr M.J. O'Hre: LCR-LON-HN5 (HN5,

2 MMUNOTHRAPY OF XNOGRAFTD HUMAN TUMOURS 255 hed nd nek), MDA-MB 468 (rest), A431 (vulvl) nd SKOV3 (ovrin). The ells were ultured routinely in Duleo's modified gle's medium (DMM) supplemented with 1% foetl lf nd serum nd ntiiotis. For estlishing xenogrfts in thymi mie, onfluent monolyers of ells were trypsinised nd resuspended t 5 x 7 ells ml- in phosphte uffered sline, ph 7.4 (PBS) just efore use. Monolonl ntiodies Rt monolonl ntiodies CR16 (gg2), CR62 (gg2) nd CR64 (gg) tht re direted ginst the externl domin of the humn reeptor for GF were prepred nd purified from sites s desried in the preeding pper (Modjthedi et l., 1993). sotype mthed monolonl ntiodies were used s ontrols nmely, ALN/l 1/53 (gg2) nd 11/16 (gg2), tht re direted ginst speifi ntigen on the rt srom HSN (Den et l., 1984) or RC/4/74 (ggl) n ntiody direted ginst n idiotopi determinnt on CRl6 (unpulished dt). ffet of mas on the growth of tumour xenogrfts in thymi mie To ssess the effet of ntiodies CRl6 nd CR62 on the growth of tumour xenogrfts two types of experiment were performed. 1. Tretment t the time of tumour inoultion 5 x 16 tumour ells in 1 jal of PBS were inoulted suutneously into oth flnks of 4 to 5 week old femle thymi (nu/nu) mie. On dy, groups of four to six mie were treted i.p. with 2 lg of CR 16, CR62 or CR64 nd further groups were treted with n equl mount of n isotype mthed ontrol or sline. Tretment ws ontinued for further 4 onseutive dys nd, therefter, three times weekly until the dy indited for eh experiment. Tumours were mesured ross two dimeters three times weekly nd the men vlues determined. When tumour regressed ompletely the men dimeter ws tken s zero for lultion of group mens ( ± s.d.). Animls were killed when the tumours rehed men dimeter of.8-1 m nd the tumours were exised, weighed then fixed in formol-sline for histologil exmintion. Animls in whih tumour growth ws ompletely or prtilly inhiited were oserved for up to 1 dys when the experiments were terminted. 2. Tretment of estlished tumours Tumour xenogrfts were set up s desried ove ut tretment ws delyed until the tumours hd rehed men dimeter of out.5 m. Unless otherwise stted, the protool for tretment with ntiody ws s efore nd ontinued until the ontrol nimls were killed. To ompre the effets of tretment on the growth of individul tumours the verge growth rte (GR) ws determined for eh tumour. Where the tumours hd regressed ompletely the results were ounted s zero nd inluded in the lultion. weight of tumour (mg) GR (mg/dy) = No of dys until exision Results Tretment with ntiody to the GFR ommening t the time of tumour inoultion HN5 tumours n the first experiment, groups of six mie were treted with ntiody CR16, ontrol ntiody ALN/ 11/53 or sline. Tretment ontinued until dy 27 when the ontrol nimls were killed euse the tumours hd rehed size of.8-1.om in dimeter. The results presented in Figure l show tht, following totl dose of 3 mg of CR16 per mouse, ll of the tumours regressed nd none were plple y dy 5. No reurrene of the tumours ws --, 1.1 ii en +1 C.1 ).1 ' Co C). Co.~._ CU F-.< Dys Figure 1 ffet on the growth of HN5 xenogrfts of tretment of thymi mie with, CR16 () or ALN/1 1/53 () from dy -27 (totl dose 3. mg/mouse);, CR62 (M) or ALN/1 1/53 () from dy -7 (totl dose 1.2 mg/mouse);, CR64 (M) or RC/4/74 () from dy -18 (totl dose 2.2 mg/dose). oserved in ny of the mie treted with CR16 nd the experiment ws terminted t dy 9. The growth of HN5 tumours ws not ffeted y tretment with the ontrol ntiody ALN/11/53 nd the results were not signifintly different from the ontrols given PBS only. Antiody CR62 inds to the sme epitope (C) s CR16 ut is of different isotype (gg2). When group of five HN5-ering thymi mie were treted with this ntiody (2pLg/tretment i.p.) no tumours were plple t seven out of the ten sites y dy 7 (Figure l) so tretment with ntiody ws disontinued (totl dose 1.2 mg). Tumours in the ontrols treted for the sme time with ALN/1 1/53 ontinued to grow s shown in Figure l. The three tumours plple t dy 7 in the CR62 treted mie regressed rpidly nd no tumour reurrene ws oserved in ny of the nimls y dy 9 when the experiment ws terminted. n third experiment, mie ering HN5 tumours were treted from the time of tumour inoultion with third ntiody, CR64 (ggl), tht is direted ginst different epitope (D) on the GFR. This ntiody, together with CR16, hd een found to e the most effetive inhiitor of C

3 256 H. MODJTAHD et l. the growth of HN5 ells in vitro. Agin, tretment with this ntiody ws effetive in using the regression of HN5 xenogrfts in mie tht hd een treted with n i.p. dose of 2 jig CR64 from dy to dy 18 (totl dose/mouse of 2.2 mg). All of the tumours hd regressed y dy 32 (Figure 1) wheres in the ontrols treted with the isotype mthed ontrol ntiody RC4/74 the tumours hd rehed men dimeter of.76 m t dy 14. We onlude tht ll three ntiodies, delivered y intrperitonel injetion, ould inhiit ompletely the growth of HN5 ells in the flnks of thymi mie when the tretment ws ommened t the time of tumour implnttion. A431, MDA-MB 468 nd SKO V 3 tumours To investigte the effet of these ntiodies on the growth in vivo of GFR overexpressing tumours of different origin, experiments were set up using thymi mie ering xenogrfts of the A43 1, MDA-MB 468 or SKOV3 tumours. t should e noted tht the SKOV3 tumour while expressing the GFR lso overexpresses the produt of the -erb-2 proto-onogene t sustntilly higher level. The totl dose of ntiody dministered to the mie in eh group ws 2.2 mg/mouse in the experiments with the A431 nd SKOV3 tumours nd.44 mg with the MDA-MB 468 tumour. n the first experiment using A431 xenogrfts, tumours were deteted t 7/8 sites y dy 3 in the ontrol nimls (Group A) nd the tumours grew progressively until dy 11 when these nimls were killed. n group C, treted with CR16 (Figure 2), tumours were plple t 6/1 sites y dy 11 nd y dy 21 tumours were growing slowly ut progressively t ll sites. As in the experiments with the HN5 tumour, ntiody CR62 (Group B) ws found to e more effetive ompred with CR16 in inhiiting the growth of A431 xenogrfts. Tumours were plple t 4/1 sites y dy 11 nd 5/1 sites y dy 21 nd when the experiment ws terminted t dy 51 four of the originl ten sites were tumour free. We onlude tht tretment from dy to dy 18 with totl dose of 2.2 mg/mouse of either CR16 or CR62 produed sustntil dely in the growth of the A431 tumour nd tht of the two ntiodies, CR62 ws sustntilly more effetive sine growth of 4/1 tumours ws ompletely prevented. n the experiments with the MDA-MB 468 xenogrfts, tumours were plple t ll sites in the three tretment groups on dy 4, ut tretment with ntiody CR62 ws prtiulrly effetive in inhiiting the growth of this tumour (Group B, Figure 3) nd ll tumours hd regressed ompletely y dy 11. All tretments with ntiody were terminted t dy 18 when totl dose of only.44 mg hd een dministered. n the CR62 tretment group, no tumour reurrene ws oserved when the experiment ws terminted t dy 1. While less effetive thn CR62, tretment t this low dose level with CR16 (Group C) indued the regression of tumours t 3/8 sites y dy 11, 4/8 sites y dy 18 nd 5/8 sites y dy 31 nd t the termintion of the experiment (Dy 1) 4/8 of the sites remined tumour free. SKOV 3 ells express sustntilly lower levels of the GFR thn do the three tumours desried ove nd none of the rt ntiodies to the GFR ws found to inhiit the growth of SKOV 3 ells in vitro (Modjthedi et l., 1993, ompnying pper). nterestingly, lthough ntiody CR16 ppered to e without effet on the growth of this tumour in vitro, tretment of the xenogrfted mie with CR62 led to smll dely in growth of the tumours (dt not shown). When the experiment ws terminted t dy 33 the men growth rtes were (± 2.64 mg/dy) for the ontrols, 8.51 (± 2.56 mg/dy) for the CR16 treted mie nd 6.38 (± 2.35 mg/dy) for the mie treted with CR62. These results indite tht tretment with ntiody CR62 delyed the development of the SKOV 3 xenogrfts. The results of these experiments showed tht the rt ntiodies to the GFR n totlly inhiit or restrit the growth of severl humn tumours tht overexpress this reeptor. Furthermore, the experiments with the MDA-MB 468 tumour suggest tht the totl dose of ntiody required to indue these effets my e sustntilly less thn tht used (3. mg) in the initil experiments with the HN5 tumour xenogrfts. m. mm mm mm w l ' L- ) ) Co o.6- S. o ).4- S.2- Dy.o- 11 n Figure 2 Growth of A431 xenogrfts in thymi mie treted from dy -18 with, ALN/1 1/53,, CR62,, CR16 (totl dose of ntiody 2.2 mg/mouse. Site without tumour (), site with tumour (), mouse killed (U, tumours >.8 m dimeter). 51

4 MMUNOTHRAPY OF XNOGRAFTD HUMAN TUMOURS mm m. No No -.6- Cu -W C.) V S S.. S OOC Dy o8 Dy o8o~ Dy 73 Figure 3 Growth of MDA-MB 468 xenogrfts in thymi mie treted from dy -18 with, ALN/11/53,, CR62, or, CR16 (totl dose.44 mg/mouse). Site without tumour (), site with tumour (), mouse killed (A, tumours >.8 m dimeter) o lw lw lw lw wwmq Dy 1 mm 1. ;.- NON. MON. mm MON. mem..8- =.4- Cu.2 Dy OU.A Figure 4 ffet of tretment with low doses of CR62 on the growth of HN5 xenogrfts. Athymi mie were treted from dy -18 with totl dose of 11 g of, ALN/1 1/53 or, CR62. Site without tumour (), site with tumour (), mouse killed (U, tumours >.8 m dimeter).

5 258 H. MODJTAHD et l. ffet of tretment with low doses of CR62 on the growth of HNS xenogrfts When mie ering xenogrfts of HN5 tumours were treted with lolg doses of CR62 from dy -18 (totl dose.11 mg) no tumours grew t 3/8 sites nd the rte of tumour growth ws restrited t the other sites ompred with ontrols treted with the sme dose of ALN/11/53 (Figure 4). These results show tht this gg2 ntiody ould ffet tumour growth t dosge whih ws some 3 fold lower thn tht used in the initil experiments with the HN5 tumour. Antiodies CR16 nd CR62 use regression of estlished tumours To investigte the effet of tretment with ntiody on the growth of estlished, progressively growing tumours, HN5 xenogrfts were set up nd the tretment with ntiody ws initited only when the tumours hd rehed men dimeter of.5 m. Figure 5 illustrtes the results otined following tretment with CR16 or ALN/1 1/53 in whih totl dose of 2.6 mg ws given s intrperitonel injetions of 2 jig from dy 9 until dy 32 when the mie in the ontrol group were killed. Soon fter the strt of tretment with CR16 growth of the HN5 xenogrfts esed nd the tumours strted to regress. The tumours hd regressed ompletely t 3/12 sites y dy 58 nd ll of the remining tumours were ontinuing to regress 45 dys fter the end of tretment with ntiody (dy 77) when the experiment ws terminted. The men weight of the tumours in the CR16 treted group t dy 77 ws 3 mg ompred with men vlue of 3 mg for the tumours in the ontrol group t dy 32. The effet of CR62 on the growth of estlished HN5 tumours is shown in Figure 6. n these experiments two groups of five mie eh were treted with totl dose of 2.2 mg of CR62 given either s five doses of 4 lsg/niml from dy 6-1 nd one of 2 pg on dy 12 (Figure 6) or s 11 doses of 2 jtg/niml from dy 6-24 (Figure 6). As ontrols, four mie were treted with 2.2 mg of ntiody 11/16 (gg2) nd two mie were treted with sline lone. Progressively growing tumours were deteted t ll sites y dy 6 nd the ontrol nimls (Group A) were killed t dy 24 when the men tumour weight ws mg (Figure 7). n the two groups treted with CR62, growth of the tumours hd esed y dy 9 nd the tumours ommened to regress. By dy 48 3/1 tumours in Group B nd 4/1 tumours in Group C hd regressed ompletely nd one further tumour in eh group hd regressed y dy 76 when the experiment ws terminted. The men weights for the tumours in Group B ws 5.17 mg nd in Group C ws 2.28 mg. We onlude tht ntiody CR62 ws very effetive in induing regression of the HN5 tumour xenogrfts sine 9/2 tumours hd regressed ompletely y the end of the experiment nd none of the tumours remining hd weight greter thn 15.8 mg (Figure 7) ompred to men tumour weight in the ontrols of 338 mg t 21 dys. There ws little differene etween the two CR62 tretment groups in either rte of tumour regression or finl result. Histologil exmintion of the tumours remining t the end of the experiment showed tht while few vile tumour ells ould e deteted numerous kertinised res were oserved suggesting tht only differentited tissues remined (dt not shown). Disussion We re prtiulrly interested in the potentil therpeuti pplition in ner ptients of ntiodies tht n inhiit growth ftor-reeptor intertion. We hve shown (Modjthedi et l., 1993 see ompnying pper) tht numer of rt ntiodies rised ginst the externl domin of the humn reeptor for GF were potent inhiitors of the growth in vitro of tumour ells tht overexpress this reeptor. n this pper we demonstrte tht three of these ntiodies, CR16 (gg2), CR62 (gg2) nd CR64 (gg1) were lso very effetive inhiitors of the growth in thymi mie of severl tumours tht overexpress the GFR. These ntiodies, whih were rised ginst the reeptor on either HN5 ells (CR16) or MDA-MB 468 ells (CR62, CR64), ll lok the inding of GF nd TGF to the GFR nd hve een shown to L.6 ~.4.2 u.uu V.WV Dys Figure 5 ffet on the growth of estlished HN5 xenogrfts of tretment of thymi mie from dy with totl dose of 2.6mg of CR16 (-) or ALN/11/53 ().

6 MMUNOTHRAPY OF XNOGRAFTD HUMAN TUMOURS (D.4.2.* Dys o * Dys Figure 6 ffet on the growth of estlished HN5 xenogrfts of tretment of thymi mie with ontrol ma 11/116 or sline (@, group A) or 2.2 mg of CR62 given s doses of:, 4 ilg from dy 6-12 (*, group B) or, 2 ilg from dy 6-24 (U, group C). inhiit the GF-indued stimultion of DNA synthesis in quiesent humn firolsts (Modjthedi et l., 1992). t is most likely tht the growth inhiition produed y these ntiodies in vitro is onsequene of loking growth ftor-reeptor intertion. Antiodies my hve n dditionl funtion in vivo euse they my e le lso to reruit nd tivte the effetor rm of the host's immune system. These funtions re dependent on ntiody isotype nd, in the rt, gg2 ntiodies re prtiulrly effetive in this respet (Dyer et l., 1989). ndeed, the results of these experiments showed tht the ntiodies ppered to e more effetive t inhiiting the growth of tumour xenogrfts thn ell prolifertion in vitro. For exmple, neither CR16 nor CR62 ould inhiit ompletely the growth of MDA-MB 468 or A431 ells in vitro nd they were without effet on SKOV 3 ells. However, in vivo CR62 ured ll the mie of the MDA-MB 468 tumours when the individul doses were only 4 jig nd the totl dose given ws.44 mg. This result is sustntilly etter thn tht reported y Mendelsohn (1989) for tretment of this tumour with ma 528 where omplete regression ws not oserved even with twie weekly doses of 2 mg/mouse. With the A431 tumour, CR62 ws not s effetive (3/8 tumours ured with totl dose of 2.2 mg) ut tretment resulted in sustntil dely in growth of the remining tumours. The ltter results do ompre fvourly, however, with those of other groups using mouse monolonl ntiodies where for exmple omplete suppression of A431 growth in thymi mie ws reported following tretment with totl dose of 12 mg of mas 225 or 528 (Msui et l., 1984). The higher doses of ntiody required to inhiit growth of

7 26 H. MODJTAHD et l C w l ldy21-3 CD 2 ioo -CA Dy 76 A B Tretment group Figure 7 Weight of tumours from experiment shown in Figure 6. Tumours were exised from ontrol mie t dy 21 ; or t dy 76 from mie treted with totl dose of 2.2mg CR62 from dy 6-12, or the A43 1 tumour my reflet the need to overome the loking effet of irulting ntigen euse this tumour is unusul in tht the ells serete lrge mounts of trunted form of the reeptor (Weer et l., 1984). ndeed, we hve found tht ulture superntnts of A43 1 ells effetively lok the inding of ll of the rt ntiodies to the reeptor for GF (Modjthedi et l., 1993). The suess of tretment lerly depends on ntigen density nd the results otined with the SKOV 3 xenogrfts suggest tht too few reeptors were present to permit effetive immune destrution. These results suggest lso tht tissues with norml levels of the GFR my suffer miniml dmge following tretment with these ntiodies. We onlude tht the greter effetiveness of the rt ntiodies in vivo is euse their tivity ws not due solely to reeptor lokde nd tht reruitment of effetor ells plyed n importnt role. Msui et l. (1986) hve lso reported enhned tivity for mouse ntiody to the GFR of the gg2 isotype (ma 528) over ma 225 (ggl) whih ound to the sme epitope nd they ttriuted the greter effetiveness to immune mehnisms involving mrophges. A similr finding hs een reported for ntiody 425 (gg2) y Rodek et l. (1987). Rt ntiodies of the gg2 isotype hve similr properties to mouse gg2 ntiodies nd some hve een shown to e prtiulrly effetive in mn in mediting ntiody dependent ellulr ytotoxiity nd tivting omplement (Hle et l., 1985; Dyer et l., 1989). These properties my lso hve ontriuted to the superior performne in the xenogrfted mie of CR62 ompred to CR16 or CR64 wheres the ltter were lerly more effetive t reeptor lokde. This spet is urrently under investigtion. CR62 indued tumour regressions with very low doses of ntiody nd totl dose of.1 1 mg led to the omplete regression of HN5 tumours t 3/8 sites. For this reson we onsider ma CR62 to e serious ndidte for linil pplition in the tretment of ner ptients with miniml residul disese where the tumours overexpress the reeptor for GF. This work ws supported y Progrmme Grnt wrded y the Cner Reserh Cmpign, London. Referenes ABOUD-PRAK,., HURWTZ,., PRAK, M.., BLLOT, F., SCHLS- SNGR, J. & SLA, M. (1988). ffiy of ntiodies to epiderml growth ftor reeptor ginst KB rinom in vitro nd in nude mie. J. Ntl. Cner nst., 8, BRADY, L.W., MYAMOTO, C., WOO, D.V., RACKOVR, M., MRCH, J., BNDR, H., DADPARVAR, S., STPLWSK, Z., KOPROWSK, H., BLACK, P., LAZZARO, B., NAR, S., MCORMACK, T., NVS, J., MARABTO, M. & SHLMAN, J. (1991). Mlignnt stroytoms treted with iodine-125 leled monolonl ntiody 425 ginst epiderml growth ftor reeptor: phse tril. nt. J. Rdition Onology Biol. Phys., 22, COWLY, G.P., SMTH, J.A. & GUSTRSON, B.A. (1986). nresed epiderml growth ftor reeptors on humn squmous rinom ell lines. Br. J. Cner, 53, DAN, C.J., STYLS, J.M., GYUR, L.A., PPPARD, J., HOBBS, S.M., JACKSON,. & HALL, J.G. (1984). The prodution of hyridoms from the gut ssoited lymphoid tissue of tumour ering rts.. Mesenteri nodes s soure of gg produing ells. Clin. xp. mmunol., 57, DRYNCK, K., GODDL, D.V., ULLRCH, A., GUTTRMAN, J.U., WLLAM, R.D., BRNGMAN, T.S. & BRGR, W.H. (1987). Synthesis of messenger RNAs for trnsforming growth ftor nd P nd the epiderml growth ftor reeptor y humn tumours. Cner Res., 47, Di MARCO,., PRC, J.H., FLMNG, T.P., KRAUS, M.H., MOL- LOY, C.J., AARONSON, S.A. & DFOR, P.P. (1989). Autorine intertion etween TGF nd the GF-reeptor: quntittive requirements for indution of the mlignnt phenotype. Onogene, 4, D MARCO,., PRC, J.H., AARONSON, S.A. & D FOR, P.P. (199). Mehnisms y whih GF reeptor nd GFo ontriute to mlignnt trnsformtion. Nt. mmun. Cell Growth Regul., 9, DVG, C.R., WST, S., KRS, M., RAL, F.X., YH, D.J., GRALLA, R., MRCHANT, B., SCHWGHART, S., UNGR, M., LARSON, S.M. & MNDLSOHN, J. (1991). Phse nd imging tril of ndium - lelled nti-gf reeptor ntiody 225 in ptients with squmous ell lung rinoms. J. Ntl. Cner nst., 83,

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