Immunotherapy and Gene Therapy of Cancer1

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1991;51:5074s-5079s.Cancer ResSteven A. RosenbergImmunotherapy and Gene Therapy of Cancer

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[ CANCER RESEARCH ( SU PPL.) 5 1. 5 07 4s -5 07 9s . S ep te mb er 1 5. 1 99 1)Immunotherapy and G ene Therapy of C ancer1S te ven A . Ros en be rgNa ti on al C anc er I ns ti tu te , B et he sd a, Ma ry la nd 2 08 92AbstractIn th e p ast d eca de, imm un oth ra pie sh ave b ee n d eve lo pe d th at arec ap ab le o f c au si ng p ro lo ng ed c an ce r r eg re ss io ns i n s el ec te d p at ie nt s w it h

advanced m etastatic disease. In the past year, attem pts at the geneth erap y o f c an cer h av e b eg un . T he se ex perim enta l c an cer tre atm en tsdeserve vigorous exploration .

The last decade has witnessed the rise of new biologicaltreatm ents for cancer based on stim ulating natural im munereactions against the disease. T hese treatm ents, collectivelyreferred to as b iolog ic al th erapy , are be gin nin g to jo in su rg ery ,radiation therapy, and chem otherapy as effective m eans fortreating cancer (1, 2). Biological therapy can be denned ascancer treatm ents that act prim arily through natural host defense mechanisms or by the administration of natural mamm alian su bstan ces. A n in creased u nderstan din g o f th e cellu larimmu ne sy stem as w ell as n ew de velopmen ts in b io tech no lo gyh av e m ad e n ew a pp ro ac he s to b io lo gic al th era py fe as ib le . U sin grecom binant D NA technology, m any biological com poundshave becom e available in large am ounts for the first tim e.O ne approach to the developm ent of biological therapies forthe treatm ent of cancer has involved attem pts to identify immune cells in tumor-bearing animals and patients that cand istin gu ish can cer from n orm al cells an d can b e u sed in ad op tiveim munotherapy. Adoptive im munotherapy can be defined asthe transfer to the tumor-bearing host of immunological reagents such as immune cells that have antitum or reactivity andc an m ed ia te , e ith er d ire ctly o r in dire ctly , a ntitumo r e ffe cts. T hem ajo r o bstacle to th e d ev elo pm ent o f effectiv e ad optiv e immunot hrapie sh as b een th e i na bil ity t o id en tif y c ells w ith s pe cif icantitum or reactivity that could be generated in large enoughn um bers for u se in can cer tre atm en t.In the past decade, however, several cell types have beenidentified that can cause the regression of cancer in somepatients. W e began our w ork in this field w ith the descriptionof LA K cells which were non-M HC-restricted killer cells cap ab le o f ly sing fresh tumo r bu t no t fresh no rm al cells in cu ltu re.T hese studies led us to the description of m ore specific antitu-m or cells called TIL that w ere capable of recognizing uniqueantigens on tum or cells. M ore recently w e have begun geneticmodification of these TIL in an attempt to increase theiran ti tumor r eact iv it y.D ev elo pm en t o f In te rle uk in 2 -b as ed Immunoth ra pie s fo rCancerThe difficulty in growing T-lymphocytes in culture for imm unological studies represented a major im pedim ent to thed ev elo pm en t o f immunoth ra pie s fo r th e tre atm en t o f c an ce r.This difficulty was largely overcome by the description of a1 Prese nted at the S ym posium . " Discoveries and O pportunitie s in C anc erR es ea rc h: A Cel eb ra tio n o f th e 5 0th A nn iv er sa ry o f t he J ou rn al C an ce r R es ea rc h, "

M ay 1 5, 1 99 1. d urin g th e 8 2n d A nn ua l M ee tin g o f th e Ame ric an A sso cia tio n fo rC an ce r R es ea rc h. H ou sto n, TX.'T he a bb re via tio ns u se d a re : LAK , l ymph ok in e- ac ti va te d k ill er ; MHC, ma jo rh is to compa ti bi li ty comp lex; T IL , t umo r- in fi lt ra ti ng l ymphocy te s; IL-2. i nt er leu -k in 2 ; T N F, tu mo r n ec ro sis fa cto r.

horm one, originally called T-cell grow th factor, which wascapable of causing the growth in culture of T-lymphocytesactivated either by specific antigen or by lectins. T his grow thfa cto r, s ub se qu en tly re name d IL -2 , o pe ne d n ew poss ib ilitie s fo rexpanding T-lym phocytes w ith antitum or activity for use inadoptive immunotherapy. In studies attem pting to isolate T -ce lls from g rowin g tum ors w e n oted th at lymp hocy tes ex po sedto IL-2 developed the ability to kill fresh tum or cells but notfre sh n orm al c ells in 4 -h c hromium re le ase a ssa ys (3 ,4 ). F urth erstudies revealed that lym phocytes from tum or-bearing as w ellas normal hosts in both the mouse and the human developedth is lympho kin e-a ctiv ate d k illin g a ctiv ity fo r fre sh c an ce r c ells.In vitro these LAK cells showed ubiquitous non-M HC-restricted k illing o f target cells altere d eith er b y m alig nant tran sfo rm atio n o r b y g rowth in cu ltu re.S ho rtly after th e descrip tio n o f th is in v itro activ ity w e b eg ane xte ns iv e s tu die s o f th e a ntitumo r e ffe cts o f th e a dm in istra tio nof IL-2 alone or IL -2 plus LAK cells (5-7). A summary of thefindings in experim ental anim als using either IL-2 alone orLAK cells plus IL-2 is shown in Tables 1 and 2. These experim en ta l s tu die s g uid ed th e d ev elo pm en t o f th e c lin ic al p ro to co lsthat followed. In these m urine studies we dem onstrated thattreatm ent with either high dose IL-2 alone or LA K cells plusIL -2 co uld m ed ia te the reg ressio n o f e stab lish ed liver an d lun gm tastasesin a v ariety o f tumo r m od els. E ach o f th ese e ffectsw as dose related and using each dose of IL-2 m ore extensivea ntitumo r e ffe cts w ere se en w ith th e c ombin ed a dm in istra tio no f LAK c ells . T he m ec ha nism o f a ctio n o f th ese immunoth era pytreatm ents was show n to depend on the activation of endogenous CD8+ cytolytic T-cells and LAK cells, as well as theex pan sio n in v iv o o f ad optiv ely tran sferred lymph ocy tes. T hu sa ba sic p rin cip le o f ado ptiv e immu nothe rap y w as estab lished inw hic h IL -2 -d ep en dent lymp ho cy tes w ith an titumo r reactivityw ere used to treat the tumo r-b earin g ho st. C ontin ue d admin istratio n o f IL -2 led to th e in v iv o ex pansio n o f th ese lymph ocy tesw hich persisted as long as IL -2 adm inistration continued. F ollow ing the discontinuance of IL -2 adm inistration these IL -2-d ep end en t antitumo r ce lls died in v iv o.O ur first studies of the in vivo adm inistration of IL-2-de-pendent cells w ere perform ed in three patients w ith advancedsarcom as w ho received cells labeled w ith either MCr or '"In tom onitor their in vivo distribution (4). It w as found that thesea do ptiv ely tra nsfe rre d IL -2 -d ep en de nt c ells d is trib ute d firs t tothe lung followed by slow clearance to the liver and spleen.A lth ou gh murin e stu die s d emon stra te d th at th e a dm in is tra tio nof both IL-2 and LAK cells w as necessary for m axim al antitum or effects, su bstan tial p rac tical d ifficu lties ex iste d in th e application of these findings to hum ans with advanced cancer.The very tiny am ounts of IL-2 that were available precludedthe adm inistration of significant am ounts to hum ans and alsoprecluded the generation of LAK cells in sufficiently largeamounts for use in human treatment. W e thus began a seriesof Phase I studies in which naturally derived IL -2 from a highproducer Jurkat cell line w as adm inistered to patients (8, 9).W e also gen erated killer cells u sin g ex posu re to p hy to hemag-glutinin and administered these cells to patients in Phase Is tud ie s (10 ).5074s

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IM MUN OTH ERAPY A ND G ENE THERA PY OF CA NCERT ab le 1 I mmun oth era py o f m urin e tu mo rs w ith I L- 2 a lo ne

1 . L iv er a nd lu ng m ic ro me ta sta se s ( 3-d ay ) fr om a v ar ie ty o f imm un og en ic a ndn on immun og en ic s ar coma s, m el an oma s, a nd a de no ca rc in oma s c an b e in hi bi ted by IL-2 admin is tra ti on.2 . L un g m ac ro me ta sta se s ( 10 -d ay ) f ro m tw o immun og en ic sa rc om as (b ut n otf rom two n on immun og en ic s ar coma s) c an b e i nh ib it ed b y I L- 2 a dm in is tr ation.3 . A d ir ec t r el at io ns hi p e xi st s b et we en th e d os ag e o f I L- 2 a nd t he ra pe uti ceffect.4 . H ig h d ose I L- 2 a dm in istra tio n le ad s to in v iv o l ym ph oid p ro lif era tio n inv is ce ra l o rg an s. T he se c ell s h av e LAK a ct iv ity in v it ro .5 . T he immu no th er ap eu tic e ff ec t o f I L- 2 o n 3 -d ay m ic romet as ta se s i s m ed ia te dby a si al o-GMi-po si ti ve LAK cel ls . I n immunogen ic t umo rs , Ly t- 2- po si ti vece ll s a l so part ic ipa te .6 . T he immun oth era pe utic e ffe ct o f IL -2 o n 1 0-d ay m ac ro me ta sta se s is m ed ia ted by Lyt -2 -p os it iv e c el ls .7 . Immun os up pr es sio n w it h i rr ad ia tio n o r c yc lo ph os ph am id e c an in hi bit I L- 2th er ap y a ga in st 3 -d ay mta sta se sb ut c an e nh an ce t he e ff ec ts o f I L- 2 o n 1 0-day macrometastases .8 . T he se ns itiv ity o f m ac ro me ta sta se s to th er ap y in vo lv in g IL -2 a pp ea rs to b ed ir ec tly r ela te d to th e e xp re ss io n o f MHC a ntig en s ( Cla ss I) o n th e tu mo r.9 . T he a dm in istra tio n o f IL -2 c an e nh an ce th e th er ap eu tic e ffe ct o f c on comi-t an tl y admi ni st er ed LAK cel ls , T IL , and s pec if ic al ly s en si ti zed T -l ympho -cytes.

T ab le 2 I mmun oth era py o f m urin e tu mo rs w ith L AK c ells p lu s I L-21 . L iv er a nd lu ng m ic ro me ta sta se s (3 -d ay ) fro m a v ar ie ty o f imm un og en ic a ndn on immun og en ic s ar coma s, m el an oma s, a nd a de no ca rc in omas c an b ei nh ib ite d b y tr ea tm e nt w it h LAK c el ls p lu s I L- 2.2 . A d ir ec t re la tio nsh ip e xists b etw ee n th er ap eu tic e ff ec t a nd th e d osa ge o f IL -2 and the dose of L AK ce lls.3 . T he p re cu rso r o f th e LAK c ell e ff ec tiv e in v iv o i s T h y-1 n eg ativ e, imm un o-g lobu lin negative, as ia lo-ASGMi posi ti ve .4 . T hre e- da y in cu ba tio n o f sp le no cy te s is o ptim al fo r th e g en er atio n o f L AKce ll s e ff ec ti ve i n v iv o.5 . Imm un oth era py o f m ic ro me ta sta se s w ith L AK c ells a nd IL -2 is e ffe ctiv e inh os ts s up pr es se d b y to ta l b od y i rr ad ia tio n o r t re atme nt w it h c yc lo ph osp ham id e. T he ra py is a ls o e ff ec tiv e i n "B" m ic e ( th yme ctom iz ed , le th al lyi rr ad ia ted, r econ st it ut ed w it h T -c el l- depl et ed bon e ma rr ow ) .6 . Imm un oth era py o f m ic ro me ta sta se s w ith a llo ge ne ic L AK c ells p lu s I L-2 iseffective.7 . L AK c ells e ffe ctiv e in immun oth era py c an b e g en era te d fro m th e sp le noc yt es o f t umor -b ea rin g m ic e.8 . M ta sta se sh at p er si st a ft er in v iv o t he ra py w ith LAK c ell s p lu s I L- 2 a res en sit iv e to LAK c ell ly sis b oth i n v itr o a nd in s ub se qu en t in v iv o e xp er im en ts . W e h av e b ee n u na ble to g en era te L AK -r esis ta nt tu mo r c ells.9 . A dm in istra tio n o f I L- 2 le ad s to in v iv o p ro life ra tio n o f tra ns fe rr ed LAKcells.1 0. D if fu se i .p . c ar cin oma to sis c an b e s uc ce ss fu lly tr ea te d w it h i. p. LAK c ell sp lu s IL-2.1 1. LAK c ell s c an media te a nti bo dy -d ep en de nt c el lu la r c yto to xi cit y; th us a dm in istra tio n o f I L-2 a lo ne o r L AK c ells p lu s IL -2 c an e nh an ce th e inv iv o t he ra pe ut ic e ff ic ac y o f mono cl on al a nt ib od ie s w it h a nt it umo r r ea ctivity.

T he cloning of the gene for IL -2, its insertion into bacteria,and th e av ailab ility o f v ery large am oun ts o f p urified recombinant IL-2, however, opened many new possibilities for thetreatm ent of patients with these approaches. Phase I studiesw ere c on du cted usin g eith er n atu ral o r recomb in an t IL -2 alo nein 3 9 p atie nts a nd u sin g e ith er p hy to hema gg lu tin in -a ctiv ate dkiller cells or LAK cells alone in 27 patients w ith advancedcancer to determ ine the safe tolerable doses of each of thesereagents (8-10). These Phase I studies have been reported indetail and served as the basis for the use of com binations of IL -2 and LAK cells in patients with advanced cancer. None of the66 patients w ith advanced cancer treated in these Phase I trialsr es ponded t o tr ea tment.In late 1984, we began studies combining treatment w ithLAK cells plus IL-2 in patients with advanced cancer and itw as with this treatm ent that w e observed our first antitum orresponses in hum ans (11-14). In accord w ith lessons learned inanimal models, LAK cells incubated for 3 or 4 days in IL-2w ere adm inistered in patients along w ith IL-2 given by bolusadministration every 8 h. An update of the results of 180patients treated as of 1990 w ith IL-2 plus LA K cells is shownin Table 3. Antitumor responses were seen in patients w ithadvanced m elanom a, renal cell cancer, colon cancer, and non-Hodgkin's lym phom a. A pproxim ately 10% of patients w ithadvanced m elanom a and renal cell cancer experienced a comp le te remis sio n o f th eir c an ce r. A s e xp erie nc e a cc umula te d w iththe use of high dose IL-2 w e began to see responses w ith IL-2administration alone and these results are summarized inT able 3.T hese stu dies demon strated th at it w as in deed po ssib le to u sestrictly im mune m anipulations to m ediate the regression ofad vance d can cer in human s. P rio r stu dies w ith a-in terfero n h addemon strated tum or reg ressio n in selec ted p atien ts w ith ren alcell cancer although the direct antiproliferative effect of a-interferon m ade it unclear as to w hether the antitum or effectsw ere seen because of an alteration in host immune reactivity orthe direct im pact of a-interferon on the growing tum or (1, 2).IL -2 itself h as n o d irect an titum or activity in an y in v itro m od ela nd th us tre atm en t w ith IL -2 o r IL -2 p lu s LAK c ells re pre se nte da true result of biological therapy in that it m ediated its anti-tumo r e ffe cts th ro ug h n atu ra l h ost d efe nse m ec ha nism s.Tumor -i nf il tr at ing Lymphocy te sA ltho ug h LAK cells co uld m ed iate antitumo r effec ts, b oth invitro and in vivo, we continued our search for more potent

T ab le 3 Immun oth er ap y o f p a tie nts w ith a dv an ce d c an ce r u si ng I L- 2 a lo ne o r w ith LAK c ell sNo . o f p at ient s

IL-2 alone IL-2 + LAK cellsCR + PRDiagnosis Evaluable" CR* PR Evaluable' CR PR CR + PRRenalMelanomaColorectalNon-Hodgkin's

lymphomaBreastSarcomaLungOtherTotal604112113111"1365000000056100000001618240000001572483071659'17884110000141763300002935211357000024

" I nc lu de s a ll tr ea te d p at ie nt s e xc ep t f ou r w ho d ie d o f th er ap y.* CR , c ompl ete r es po ns e; PR , p ar ti al r es po ns e.' In cl ud es a ll t re at ed p ati en ts e xc ep t o ne l os t t o f oll ow -u p a nd o ne w ho d ie d o f th er ap y.Two p atie nts e ac h w ith h ep ato ma a nd b ra in c an ce r; o ne e ac h w ith o va ry , p an cr ea s, a nd u te rin e c an ce r.' O ne p ati en t e ac h w ith c an ce r o f b ra in , e so ph ag us , o va ry , t es te s, th yr oid , g as tr in oma . u nk nown p rima ry a nd two p at ie nt s w ith H od gk in 's lymph oma .

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IM MU NOT HERAPY A ND GE NE TH ERAPY OF CANCE Rlym phocytes that m ight be used in adoptive immunotherapy.O ur sp ecific in terest w as th e iden tificatio n of T -ce lls th at c ou ldre co gn iz e s pe cific tumo r a ntig en s in a MHC-re stric te d fa sh io n.Techniques were developed for isolating TIL from resectedtumors and these cells provided evidence of specific tumorantigen recognition by lym phocytes obtained from a tum or-bearing host (15-18). These cells were isolated by culturingsingle cell suspensions from tum ors in IL -2. Infiltrating lymp hocy te s th at b ore IL -2 recep to rs, p resumab ly b ecau se o f th eirreactivity to tumo r, g re w in th e p resen ce o f IL -2 an d d estro yedtum or cells sim ultaneously grow ing in the culture. T hus, 2 to 3w eeks after initiating cultures pure populations of T IL couldbe established without the presence of contam inating tum orcells. W e established techniques for isolating TIL from bothm ouse and hum an tum ors and extensively studied these cellsin vitro as well as in tumor models in m ice. In the mouse, TILce lls w ere ex clu siv ely CD8 + alth oug h in the human bo th CD4 +and CD8+ T-cells grew in culture. In the mouse, specificrecognition of tum or antigens could be identified on the basisof specific lysis of target cells as w ell as by specific cytokinesecretion when TIL w ere coincubated w ith the type of tum orce lls from w hich th ey w ere d erive d (1 9-2 1). T hese reactivitiesw ere M HC restricted and could be inhibited by m onoclonala ntib od ie s a ga in st C la ss I a ntig en s.Extensive studies were conducted in anim al m odels of theantitum or effects of tum or-infiltrating lym phocytes. T hesestudies are summarized in Table 4. T itration of TIL in micew ith e sta blish ed lu ng a nd liv er mta sta se sin dic ate d th at th es ecells were from 50 to 100 times more potent than LAK cells inre du cin g lu ng mta sta se s(1 5). In a dv an ce d tumo r mod els, T ILplus IL-2, but not LAK cells plus IL-2 in conjunction withc yc lo ph osp hamid e, w as e ffe ctiv e in e lim in atin g e sta blish ed mtastases from the lung and liver.F ollow ing these early studies w ith T IL cells, attem pts w erem ade to find w ays to im prove their antitum or efficacy in m ice.B ecause of our finding that T IL m ight not be effective againsttumors that expressed very low levels of Class I antigens wetransfected genes coding for Class I antigens into tum ors andshow ed that T IL raised from these tum ors could m ediate theirregression as w ell as the regression of the parental non-Class Iexpressing tum or after treatm ent of the m ouse with -y-inter-feron. Gamma interferon was shown to up-regulate Class IM HC antigens in vivo and thus the vital role played by M HCex pressio n in th ese tum or immu no th rapies w as establish ed(22-26). W e further show ed that local tum or irradiation could

T ab le 4 I mmun oth era py o f m urin e tu mo rs w ith T IL a nd IL -21. T he adm inistration o f T IL + IL -2 is 50-100 tim es m ore e ffec tive than L AKc el ls + I L- 2 in r ed uc in g e sta bli sh ed ( da y 3 ) l un g m ic romet as ta se s.2 . T he a dm in istr atio n o f T IL + IL -2 + c yc lo ph osp hamid e is e ffe ctiv e in tre atin g m ic e w ith a dv an ce d lu ng ( da y 1 4) o r l iv er ( da y 8 ) m ta sta se s.Al l threeagent s a re r equi red for e ff ec ti ve t re a tment .3. T he phenotype of TIL e ffec tiv e in vivo is C D3+ , C D4-, C D8+ .4 . T IL ra is ed fro m tumo rs th at e xp re ss C la ss 1 a ntig en s o nly a fte r tra nsf ec tio nw ith g en es c od in g f or C la ss I c an med ia te th e r eg re ss io n o f m ic rometa sta se sf rom th e p ar en ta l tumor a fte r t re atme nt o f th e mo us e w ith - y- in te rf er on .5 . L oc al tu mo r irra dia tio n sy ne rg iz es w ith T IL a nd IL -2 a dm in istra tio n inmedi at in g t he r eg re ss io n o f e st ab li sh ed mta st as es .Lo ca l i rr ad ia ti on cansubs ti tu te fo r cyclophosphamide.6 . T IL w ith im pro ve d a ntitu mo r a ctiv ity in v iv o c an b e g en era te d fro m tumo rs us pe ns io ns b y u si ng immun omagn et ic b ea ds t o is ola te l ymp ho cy te s f ol lowe db y in cu ba tio n o f lymph oc yt es in low d os e I L- 2.7 . I n m ic e c ure d o f lu ng m ic ro me ta sta se s b y a dm in istra tio n o f T IL . liv e T I Lc an b e i de nt if ie d in v iv o 3 mo nth s a ft er in je cti on .8 . T he sp ec ific s ec re tio n o f f -in te rf ero n b y T I L w he n c ultu re d w ith tu mo r is th eb es t in v itr o c or re la te o f th e i n v iv o a nti tumor e ff ec ti ve ne ss o f T IL . N on ly ti cT IL t ha t s peci fi ca ll y s ec re te > -i nt er fe ro n can e ff ec ti ve ly t re at e st ab li sh ed l ung

T ab le 5 T IL tr ea tm en t o f p a ti en ts w ith me la noma "

NR "Objectiveresponse(PR + CR) PR + CR(all)( numbe r o f p a ti en ts )No p revi ou sL-2IL-2+YIL-2(N oY)Previous

IL-2IL-2+YrIL-2(no CY)177341143111/284/113/61/539365020

micrometastases.

" E xc lu de s p at ie nts w ith b ra in mt as ta se s t s ta rt o f t re atme nt ( al l NR ).* N R , n o r esp on se ; P R, p artia l re sp on se ; CR, c om ple te re sp on se ; CY, c yc lophosphamide.' E xc lu de s tw o p atie nts w ho re ce iv ed IL -2 a t 3 0,0 00 /k g; b oth w ere NR.

substitute for cyclophospham ide in synergizing w ith T IL andIL-2 (27). The reason for the need for this local tumor treatm en t, eith er w ith cy clo ph osp ham ide o r irrad iatio n is n ot cle ar.It is possible that these treatments result in elim ination ofsuppressor cells or increase the traffic of im mune cells to thetum or site. The cytoreductive effect of these treatm ents m aya lso p la y a ro le in th eir e ffe ctiv en ess .M ore recen tly, im pro ved tech niq ues fo r gen erating T IL w ithantitumo r activ ity in v iv o h ave b een estab lished . T he u se o f lo wdose IL -2 (60 to 120 lU /m l) generates cells w ith m ore specificcytolytic a ctiv ity a nd m ore effectiv en ess in v iv o th an th e u se o fhigh dose IL-2 (6000 lU /m l) (21). The long term persistenceof TIL in mice cured by the in vivo injection of TIL has beendemonstrated using congenie Thy-1.1 TIL in Thy-1.2 miceb ea rin g sy ng en eic tumo rs (2 8). E xte ns iv e e ffo rts to id en tify th echaracteristics of TIL in m ice that correlate w ith antitum orreactivity have show n that specific cytokine secretion ratherthan specific lysis is m ost critical (20). Thus these studies ofthe in vivo antitumor activity of TIL in murine models haveplayed an im portant role in guiding the design and conduct ofhum an trials w ith T IL in patients w ith advanced cancer.Based on these anim al m odels, pilot studies have begun thetre atm en t o f p atie nts w ith a dv an ce d m eta sta tic m ela noma u sin gtum or-in filtrating lym ph ocytes (29 ). T he results o f o ur first 5 0patients w ith advanced m elanom a treated w ith T IL are show nin T able 5. T hirty-eight % of patients have responded to treatm en t. In te re stin gly , sim ila r re sp on se ra te s w ere se en in p atie ntspreviously failing high dose IL -2-based therapy com pared tothose that had previously not been treated w ith IL -2.T IL h av e b een v alu ab le fo r stu dy in g th e n atu re o f the immun eresponse of hum ans to their cancers. From approxim ately one-third of patients w ith m elanom a, T IL w ith specific cytolyticactivity for the autologous tum or and not autologous norm altissues can be isolated (16-18). M ore recently w e have show nthat specific cytokine secretion can also identify im mune res po nse s in p atie nts w ith g rowin g m alig na nc y. S pe cific c yto kin es ec re tio n h as id en tifie d immun e c ells in p atie nts w ith m ela nomaa nd b re as t c an ce r.TIL have been valuable in studying the nature of sharedantigens am ong patients with cancers of sim ilar histology.E xtensive studies of T IL from patients w ith m elanom a testedagainst a panel of allogeneic melanomas with known HLAsubtypes have dem onstrated that m elanom as from differentindividuals appear to share com mon tum or antigens that arerestricted by a specific HLA specificity (30). The HLA-A2genotype appears to be particularly com mon as a restrictionelem ent in the recognition of melanom a antigens. To furtherdem onstrate the nature of the shared antigens in patients w ithmelanoma, we have transfected the gene for HLA-A2 into

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IM MUNO THE RA PY A ND GE NE T HERAPY OF CANCE Rmelanomas and have shown that 6 of 6 different melanomastransfected with the gene for HLA-A2 are recognized by asingle H LA -A 2-restricted T IL (31). It thus appears that antigens shared among melanomas can be recognized by TIL andpossibilities for the use of these antigens for immunization ofpatients against cancer appear feasible. U sing T IL as a recognition system we are currently in the process of attem pting toclone the gene that codes for tum or antigens in both the m ouseand hum an by transfection of genes from sensitive to resistantcell lines. S hould such genes be cloned they could be insertedinto vaccinia or other viruses for use in the immunization ofp atie nt s a ga in st c an ce r a nt ig en s.F inally studies w ith T IL have dem onstrated that these cellsrecirculate and specifically localize in cancer deposits. U p to0.015% of all injected TIL localized per g of tum or based on invivo studies in humans using TIL labeled with '"In (32, 33).T hus T IL cells have proved to be a valuable resource not onlyfor the study of the im mune reaction of patients against theirtum ors but also for use in im munotherapy. Extensive effortsunder w ay to im prove upon immunotherapy w ith T IL are listedin T able 6 including attem pts to enhance the in vivo effectiveness of existing TIL or to generate m ore effective TIL eitherb y g rowin g m ore effectiv e cells, b y g en etically m od ifying T IL ,o r b y raisin g T IL from g en etically m od ified tumor. In ad ditionstud ies are u nder w ay to stu dy th ese IL -2 -b ased immu no th rap ie s in c on ju nc tio n w ith o th er tre atm en ts su ch a s c hemo th era pyand radiation therapy. N ew recom binant cytokines are beingtested fo r an titumo r reac tiv ity . W e h ave recen tly p ub lish ed o urresults show ing that IL -6 has antitum or activity w hen adm inistered to tumo r-b earin g m ice (34 ) a nd clin ical tria ls w ith IL -6w ill b e u nd erta ke n sh ortly .

Gene Therapy of CancerThe ability to introduce and express foreign genes in eu-karyotic cells has opened new possibilities for the therapy ofcan cer. O ur first attem pts at the g en e th erapy of can cer in vo lv ed

m odification of T IL by the insertion of m arker genes (35, 36).Mo re re ce ntly g en es c od in g fo r c yto kin es h av e b ee n in tro du ce din to T IL .In the first phase of these studies we inserted into TIL aba cterial g en e cod in g fo r n eom ycin p hosp ho tran sferase w hichcould induce resistance to the antibiotic neom ycin and enableu s to d iffe re ntia te a do ptiv ely tra ns fe rre d T IL from e nd og en ou shost lymphocytes (35, 36). The goal of these studies was todem onstrate the feasibility and safety of using retroviral m ediated gene transfer to introduce genes into humans and tostu dy th e lo ng term d istribu tion and su rviv al o f au to lo gou s T IL .B ecau se o f th e p ractical, safe ty , an d e th ical issu es in vo lv edwith human gene transfer and the possible consequences ofusing a m odified retrovirus derived from the M oloney m urineleukem ia retrovirus random ly integrating a new gene into theh um an g en om e, o ur clin ic al stu dies utilizing h um an T IL tran sduced with the gene coding for neom ycin resistance (NeoR)w ere p re ce de d b y e xte nsiv e in v itro stu die s a nd w ere e xte ns iv elyreview ed b y a v ariety o f rev iew g ro up s compo sed o f clin icia ns,scientists, ethicists, and lay people. In these studies w e demonstrated that the gene could be successfully inserted intohum an T IL , that the properties of hum an T IL w ere not altered,that the gene w as expressed in T IL , and that these procedurescould be perform ed with little risk to the patient and no risk toh ealth care perso nn el an d the pu blic. R etro viral m ed iate d gen etransd uctio n of T IL w as selected b ecau se o f th e h ig h efficienc y

Tabl e 6 Expe rimen ta l l eads f or improv in g immunot he rapy\. E nh an cin g in v iv o e ff ec ti ve ne ss o f T ILa . C ombi na ti on w ith o th er c yto ki ne sb . C omb in ati on w it h l oc al r ad ia ti on th er ap y2 . G en er at in g mo re e ff ec tiv e T ILa . L ymph oc yte s ub po pu la ti on s o r c lo ne sb . Repeat ed i n v it ro s timul at io nc . L ow -d ose IL -2d. Culture in IL-2 + IL-4e . M od ify T IL b y g e ne tra ns fe r3 . Immun iz e w ith g en e-mo dif ie d tumora . C yto ki ne g en esb. MHC antigen genes4 . A pp li ca ti on o f n ew c yto ki ne sa. IL-6b . Cy to ki ne comb in at io ns5 . Monocl on al ant ib od ie sa . + IL -2 + LAK c ells ( LAK c ells m ed ia te a ntib od y-d ep en de ntb . + macrophage -co lo ny -s timul at in g f ac to r6 . S yn erg y o f c hemo th er ap y 4 - I L- 2

o f ge ne tran sfer u sing th is tec hn iq ue (3 7, 38 ).Studies perform ed on TIL from m ultiple cancer patients aswell as the 10 patients who have now received NeoR gene-modified TIL demonstrated that the NeoR gene could be inserted into h um an T IL , th at it w as satisfacto rily ex pressed , an dthat the general properties of the TIL were not changed (35,36). Sem iquantitative Southern blots indicated that approxim ately one viral genom e copy w as present in the gene-transduced T IL . T he phenotype and cytotoxicity of the transduceda nd n on tra ns du ce d c ells w ere s im ila r. A na ly se s o f T -c ell re ce pto r g en e rearran gem en ts reve aled th e o ligo clo nal n atu re of th eTIL population and no major changes in the DNA rearrangem ent patterns or the level of m RN A expression of the and 7 chains follow ing transduction and selection of TIL inthe neomy cin ana lo gu e G 418 (35 ). S im ilarly , cytok in e mRNAexp re ssio n w as n ot sig nific an tly a lte re d fo llowin g th e tra nsd uctio n o f T IL .Transduced TIL consistently show ed resistance to G418, an eomy cin an alo gu e letha l to all e uk ary otic ce lls. T he p olymer-ase chain reaction technique was utilized to detect the N eoRge nome. A pp rox im ately o ne tran sd uced cell in 1 0s n orm al cellsc ou ld b e c on sis te ntly d ete cte d (3 6).S afety co nsid eratio ns w ere p aram oun t in o ur co nsid eratio nof the use of this gene transduction technique for introducinggenes into TIL for use in patient therapy. Extensive safetystudies w ere perform ed on all transduced T IL including testsfo r aero bic a nd an aerob ic b acte ria, fu ng i, an d Myco plasm a an dte sts u sin g s arc om a-p ositiv e, le uk em ia -n eg ativ e a ss ay s fo r e co -tro pic , x en otro pic , a nd ampho tro pic in fe ctio us v iru se s. Amplification tests using NIH 3T3 cells were performed and wereshow n to be capable of detecting a single viral particle per m lof solution. All safety tests perform ed on TIL in preclinicalstudies as well as on TIL from the patients who received thegene-m odified cells w ere negative and no safety problem s ofany kind were detected (36). Gene-modified cells could bed etected in th e circu lation u p to 18 9 d ay s an d in tumor d ep ositsup to 64 days after the infusion of gene-m odified T IL (36).L aboratory and clinical studies using T IL transduced w ithth e g en e fo r n eomycin resistan ce d em on strated th at retro viralm ediated gene transduction w as a feasible and safe m eans forin tro ducing ge nes in to h um an s. B ecau se o f th e accumu latio n o fT IL at tum or deposits w e have conducted studies attem ptingto m odify hum an TIL with genes that can im prove the antitu-m or effectiveness of these cells. The first gene that w e haveselected for these studies is the gene for T NF .E xtensive anim al research in the S urgery Branch, N ationalC an cer In stitu te, an d in m an y othe r g ro up s h ave d em onstrated5077s



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th at the in jection o f reco m bin an t T NF can m e diate th e nec ro sisan d reg ressio n of a v ariety o f estab lish ed m u rin e c anc ers. H owev er, tum or-bearing m ice can tolerate up to 400 M g/k g T NFand these doses are required to m ediate tum or regression; theadm in istratio n o f less T NF is f ar less ef fectiv e. In co ntrast, them ax im um tolerated dose of T NF in hum ans in both S urgeryB ran ch , N atio nal C an ce r In stitu te , an d o th er s tu die s is ap pro ximate ly 8 / g/k gd ay O )- T h us wh en g iv en i.v . in je ctio ns h uman scan tolerate only 2% of the TN F dose required to m ediateantitum or ef fects in the m ouse and this appears to ex plain w hyantitum or ef f ects hav e not been seen w hen T N F w as adm iniste re d s ystem ic ally to h uman s.W e hav e thus sought m eans to selectiv ely increase the localconcentration of T N F at the tum or site. B ecause T IL can traf f icdirectly to tum or deposits and concentrate at those sites (32,3 3) w e h av e h yp oth esiz ed that T IL th at are g en etically m o dif iedto produce large am ounts of TN F m ay generate high TN Fconcentrations in the local tum or m icroenv ironm ent and thusex hib it an increased an titum o r ef fect co m pared to no rm al T IL .T he retrov iral v ector construct selected f or insertion of theT NF gene into hum an T IL contains the T NF gene prom otedb y th e m u rin e lo ng term inal re peat and th e ne om y cin resistancegene prom oted by the S V 40 early prom oter region. T he tw o-gene retrov iral construct w as introduced into the PA 3 17 producer cell line and the supernatant f rom this line w as used totransduce the T IL from patients w ith adv anced m etastaticmelanoma.A l th ou gh it h as b een relativ ely easy to ex press cy tok in e ge nesin tum or cells and dem onstrate production of large am ounts ofT N F, dif f iculties ex ist in the ex pression of cy tok ine genes inly m phocy tes. It has been dif f icult to achiev e consistently highlev els of cy to kin e p rod uctio n in ly m ph ocy tes usin g these retro -v iral v ectors p ro bably b ecau se reg ulato ry m e chanism s ex ist f orc yto kin e tran sc rip tio n, tran slatio n, an d/o r s ec re tio n in lymp hocy tes that do not ex ist in other cell types. W e have, how ever,been able to achiev e the expression of T NF genes in lym phocy tes f ro m selected p atien ts an d h av e ac hiev ed cy to kin e secretion in ex cess of 100 pg/106 cells/24 h.W e hav e begun clinical studies using these T N F gene-m odif ied T IL in p atients w ith adv an ced cance r. In creasin g n um b ersof T NF-transduced T IL are being adm inistered, f irst in theabsence of IL -2 and then w hen safely tolerated doses areachieved, in conjunction w ith IL -2. T o the present tim e fourpatients w ith adv anced m elanom a hav e been treated w ith theT N F gene-m odif ied T IL . N o side ef f ects hav e been seen at celldoses w hich hav e ranged up to 10" cells in a single infusion. Alarg e series o f patien ts treated w ith g en e-m o dif ie d T IL plus IL -2 w ill be required to k now w hether or not this approach giv esim prov ed results com pared to the use of unm odif ied T IL . W ecurrently hav e perm ission to treat up to 50 patients w ith T N Fg en e-mo dif ie d T IL u tiliz in g th is ap pro ac h.A dditional genes being studied for insertion into T IL toim p rov e th eir an titu m or activ ity in clu de 7 -in terf ero n, IL -2, IL -6 , an d g en es f or c him e ric T -c ell re ce pto rs .M ore recently w e hav e begun studies inv estigating the use ofgenetic m odif ication of tum or cells to increase their im m uno-genicity . W e and others hav e dem onstrated that insertion ofcy tok ine genes can increase the imm une recognition of tum orcells and can lead to the production by the host of cy toly ticcells that are not produced in response to the parental non-m odif ied tum or (39-43). T hese ex perim ents hav e been conducted by a v ariety of groups and include insertion of genes forIL -4, IL -2, tum or necrosis factor, 7-interf eron, and granulo-

c yte -mac ro ph ag e-c olo ny -s tim u latin g f ac to r. In re late d e xp erim ents w e hav e show n that insertion of the gene for Class Im ajo r h isto co m patib ility an tigen s can also in crease th e imm u -nogenicity of tum ors and lead to the generation of T IL thatcannot be produced f rom low M HC-ex pressing tum ors (26).T h is ap pro ac h to g en e th erap y , b y g en etic ally mo dif y in g tumorsfor use in im munization, holds prom ise not only for activ eim m unotherapy but also for the dev elopm ent of im m une cellsth at m ig ht be u se d in ad op tiv e imm u no th erap y.In the last decade biological therapy has been show n to becapable of m ediating the regression of established cancers inso m e p atien ts w ith ad van ced m align an cy . T he co ntin ued d ev elo pm e nt o f these b io lo gical app ro ach es o ff ers th e h op e that th eycan lead to the dev elopm ent of ef f ectiv e, safe, and practicaltre atm e nts f or p atie nts w i th c an ce r.References1 . R o se nb erg , S . A . . L o ng o. D . L ., an d L o tz e. M . T . Pri nc ip le s an d ap pli cat io nso f b io lo gi c t he rap y . I n: V . T . D e V i ta, S . H ell m an , an d S . A . R o se nb erg ( ed s.) .C an ce r, Pri nc ip le s an d Prac tic es o f O nc ol og y, E d. 3 , p p. 1 34 2-1 39 8. Ph ilad elp hi a: J. B . L ip pin co tt C o., 1 98 9.2. D eV ita, V ., H ellm an, S ., and R osenberg, S . A . (eds.). B iologic T herapy ofC an ce r. Ph ilad el ph ia: J. B . L ip pin co tt C o., 1 99 1.3. Y ron, !.. W o od. T . A ., S piess, P. J., and R osenberg, S . A . In v itro grow th of

m u rin e T cells. V . T he iso latio n an d g ro wth o f ly m ph oid cells in filtratin gs yn ge ne ic s oli d t um o rs . J. I mm un ol., 1 25 : 2 38 -2 45 , 1 98 0.4. L otz e, M . T ., L ine, B . R .. M athisen. D . J., and R osenberg, S . A . T he in v iv od is tri bu ti on o f au to lo go us h um an an d m u rin e ly m p ho id c ell s g ro w n in T c ellgrow th f actor (T CG F): im plications f or the ad optiv e im m unotherapy oft um o rs . J. Imm u no l.. 1 25 : 1 48 7-1 49 3, 1 98 0.5. L af reniere. R ., and R osenberg, S . A . A d optiv e im m unotherapy of m urineh ep at ic m t as tas esw i th l ymphok in e ac ti v at ed k i ll er ( L AK ) c el ls an d re comb in an t i nt erl eu k in -2 ( R IL - 2) c an me di at e t he re gre ss io n o f b ot h immunoge ni can d n on -imm u no ge nic s arc om as an d an ad en oc arc in om a. J. Imm u no l., 1 35 :4 27 3-4 28 0, 1 98 5.6. Papa, M . Z ., M ule, J. J., and R osenberg, S . A . T he anti-tum or ef f icacy ofl ymphok i ne -a ct iv at ed k i ll er c el ls an d re comb in an t i nt erl eu k in -2 i n v i vo : imm u no ge ni c an d n on imm u no ge ni c m u ri ne t um o rs o f t hre e d is ti nc t h is to lo gi et yp es . C an ce r R e s.. 4 6: 4 97 3-4 97 8. 1 98 6.7. M ule, J. J., V ang, J. C., L afreniere, R .. S hu, S .. and R osenberg, S . A .I den ti f ic at ion o f c el lu l ar mechan i sms opera ti onal i n v i v oduring the reg re s si ono f e st ab li sh ed p ulmonary m t as tas esb y t he s y st em i c adm in is trat io n o f h ig h-d os e re com bi nan t in te rl eu ki n-2 . J. Imm u no l., 1 39 : 2 85 -2 94 . 1 98 7.8. L otz e, M . T ., Frana, L . W ., S harrow , S . O .. R obb. R . J., and R osenberg , S .A . In v iv o ad m in istratio n o f p urif ied h um an in terleu kin -2 . 1 . H alf lif e an dim m u no lo gie ef fects o f th e Ju rk at c ell lin e d eriv ed IL -2 . J. Im m un ol., 1 34 :1 5 7- 16 6, 1 98 5 .9. L otz e, M . T ., M atory , Y . L ., E ttinghausen, S . E ., R ay ner, A . A ., S harrow ,S . O ., S eip p, C . A ., C us te r, M . C ., an d R o se nb erg , S . A . I n riv o adm in is trat io no f p urif ied h um an in te rleu kin -2 . II. H alf lif e an d im m un olo gie ef fects an dex pan sio n o f p erip he ral ly m ph oid cells in v iv o w ith reco m bin an t IL -2 . J.Imm u no l., 1 35 : 2 86 5-2 87 5, 1 98 5.1 0. R o se nb erg , S . A . Imm u no th erap y o f c an ce r b y t he s ys te m ic adm in is trati ono f ly m p ho id c el ls p lu s i nte rle uk in -2 . J. B i ol. R e sp on se M o di f., 3 : 5 01 -5 11 ,1984.11. R osenberg. S . A ., L otz e, M . T ., M uul, L . M ., L eitm an, S ., Chang, A . E.,E ttin gh au sen , S . E ., M ato ry , Y . L .. S k ib ber. J. M ., S hilo ni, E ., S eip p, C . A . ,S im p so n, C ., an d R e ic he rt , C . M . O bs erv ati on s o n t he s ys te m ic adm in is trati on o f au to lo go us ly m p ho kin e-ac tiv at ed k ill er c ell s an d re com bi nan t in te rleu kin -2 in p atien ts w ith m etastatic can cer. N . E ng l. J. M e d.. 3 13 : 1 48 5-1 49 2, 1 98 5.12. R osenberg, S . A ., L otz e, M . T ., M uul, L . M ., Chang. A . E.. A v is, F. P.,L eitm an, S ., L inehan, W . M ., R obertson, C . N ., L ee, R . E., R ubin, J. T .,S eipp. C . A .. S im pson, C . G ., and W hite. D . E. A progress report on thetre atm e nt o f 1 57 p ati en ts w ith ad v an ce d c an ce r u sin g ly m p ho ki ne ac tiv at edk iller c ells an d in terleu kin -2 o r h ig h d ose in terleu kin -2 alo ne. N . E ng l. J.M e d., 3 16 : 8 89 -9 05 . 1 98 7.13 . R osenberg, S . A . Im m unotherapy of patients w ith adv anced cancer usingi nt erl eu k in -2 al on e o r i n c omb in at io n w i th l ymphok i ne ac ti v at ed k il le r c el ls .In: V. D eVita , S. Hellman. a nd S. A. Ro se nbe rg ( eds .) . Impo rta nt Adva nc esi n O nc olo gy , p p. 2 17 -2 57 . Ph il ad el ph ia: J. B . L ip pi nc ott C o., 1 98 8.14. R osenberg. S . A ., L otz e, M . T .. Y ang, J. C .. A ebersold, P. A ., L inehan, W .M ., S eipp, C . A ., and W hite, D . E. Ex perience w ith the use of high-dosein terleu kin -2 in th e treatm en t o f 6 52 p atien ts w ith can ce r. A n n. S urg ., 2 10 :4 74 -4 85 , 1 98 9.15. R osenberg. S . A .. S piess, P., and L afreniere. R . A new approach to thea do pt iv e immuno th erap y o f c an ce r w i th t umo r- in f il trat in g l ymphoc y te s. S c ie nc e ( W as hi ng to n DC) . 2 23 : 1 21 8-1 32 1. 1 98 6.1 6. M u ul , L . M . , S p ie ss , P. J., D ire cto r, E . P., an d R o se nb erg , S . A . I de nti fi cat io n

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IM MUNO THE RA PY A ND G ENE T HERAPY OF CAN CERo f s pe cif ic c yto ly tic immun e r es po ns es a ga in st a uto lo go us t umo r i n h uman sb ea rin g m alig na nt m ela noma . J . Imm un ol., 1 04 : 3 66 -3 76 , 1 98 7.1 7. T op alia n, S . L .. M uu l. L . M ., S olo mo n. D .. a nd R ose nb er g. S . A . E xp an sio no f h uman t umo r in fil tr ati ng lymph oc yte s f or u se in immun oth er ap y t ri als . J .Imm un ol. M eth od s, 1 02 : 1 27 -1 41 , 1 98 7.1 8. T op ali an , S . L ., S ol omon , D ., a nd Ros en be rg , S . A ., T umo r- sp ec if ic c yto ly si sby lym phocytes infiltrating hum an m elanom as. J. Immunol., 142: 3714-2725, 1 989. "

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