REGRESSION OF A DISSEMINATED SOLID TUMOR BY SYSTEMIC TRANSFER OF LYMPHOID CELLS EXPANDED IN INTERLEUKIN 2

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1 REGRESSON OF A DSSEMNATED SYNGENEC SOLD TUMOR BY SYSTEMC TRANSFER OF LYMPHOD CELLS EXPANDED N NTERLEUKN 2 BY TMOTHY J. EBERLEN, MAURY ROSENSTEN, AND STEVEN A. ROSENBERG From the Surgery Branch, Dvson of Cancer Treatment, Natonal Cancer nsttute, Bethesda, Maryland Specfc adoptve mmunotherapy s a theoretcally attractve approach to the treatment of tumors, although few examples exst of the effectve treatment of establshed syngenec sold tumors by ths approach (1). Early reports descrbed the use of large numbers of thoracc duct lymphocytes from mmunzed anmals (2) as well as lymphocytes from mmunzed allogenec and xenogenec anmals (3) n an attempt to eradcate sold tumors. Borberg et al. (4) treated Meth A sarcomas wth up to mmunzed syngenec lymphocytes and succeeded n causng regresson of establshed tumors. Usng the Meth A tumor, but an alternatve method of mmunzaton, Berendt and North (5) demonstrated that the ntravenous nfuson of senstzed T cells from mmune donors could cause complete regresson of large establshed tumors growng n T cell-defcent hosts. They also showed that nfuson of splenc T cells from tumor-bearng donors could nhbt ths regresson of establshed tumor, suggestng that falure to reject ths tumor was the result of suppressor T cells n the tumor-bearng host. Fernandez-Cruz et al. (6) showed that ntravenous nfuson of mmune lymphocytes was capable of curng rradated rats bearng a subcutaneous tumor. A common factor n these adoptve mmunotherapy nvestgatons was the large number of senstzed lymphocytes that were requred to cause the regresson of establshed sold tumors. Because the expanson of lymphod cells n nterleukn 2 (L-2) 1 s capable of generatng large numbers of specfcally mmune cells, we have explored the possble use of these cells for the adoptve mmunotherapy of a sold tumor. T lymphocytes expanded n L-2 retan cytotoxc as well as other functonal mmunologc propertes, and ndvdual lymphocytes can he cloned to large cell numbers wthout loss of mmunologc specfcty (7-11). Usng these technques, we have demonstrated that specfcally senstzed cells expanded n lectn-free L-2 (LF-L-2) were capable of medatng allogenec skn graft rejecton (12) when adoptvely transferred to normal hosts. More recently, we have establshed long-term T lymphod lnes specfcally lytc for the syngenec FBL-3 lymphoma (13) and have successfully used these cels n the adoptve chemommunotherapy of a dssemnated mcrometastatc tumor (14). 1 Abbrevatons used n ths paper: CM, condtoned medum; Con A, concanavaln A; FCS, fetal calf serum; HBSS, Hanks' balanced salt soluton; L-2, nterleukn 2; VS, n vtro senstzaton; LF, lectn-free; LSM, lymphocyte separaton medum. Journal of Expermental Medcne Volume 156, August

2 386 REGRESSON OF A DSSEMNATED SYNGENEC SOLD TUMOR n ths paper we have extended these observatons to the treatment of an establshed palpable syngenec tumor n the footpad of C57BL/6 mce at a tme when the tumor s also dssemnated throughout the host. We have demonstrated that mmune lymphocytes, n vtro senstzed and expanded n LF-L-2 as a sole treatment, are effectve n curng mce of both local tumors and dssemnated metastases after one ntravenous adoptve transfer. Materals and Methods Anmals. 16-wk-old C57BL/6 mce were used n these experments. They were obtaned from The Jackson Laboratory, Bar Harbor, ME. Tumors. FBL-3 (kndly suppled to us by Dr. C. C. Tng, Natonal Cancer nsttute) s a Frend vrus-nduced lymphoma/leukema transplanted n C57BL/6 mce n ts asctc form. Ths tumor bears tumor specfc and/or vral antgens that cross-react wth other tumors nduced by the Frend, Moloney, and Rauscher vruses (15) and grows well n the asctc form n syngenec mce. Ths tumor wll grow and regress n a normal C57BL/6 mouse f njected ntramuscularly or subcutaneously, although t grows progressvely at these stes n rradated mce. MCA-103 s a methylcholanthrene-nduced fbrosarcoma nduced n C57BL/6 mce n our laboratory as prevously descrbed (16). t was mantaned by seral ntramuscular passage. n Vvo mmunzaton wk-old C57BL/6 female mce were mmunzed wth one ntramuscular njecton of 107 lve FBL-3 suspended n 0.05 ml sterle Hanks' balanced salt soluton (HBSS). An ntramuscular tumor wll grow for ~2 wk and then completely regress. All anmals used as mmune spleen donors had no evdence of tumor at the tme of spleen harvest. Less than 10% of the mmunzed anmals grew tumors that resulted n the death of the anmal. n Vvo Assay of Adoptve lmmunotherapy. Mce were gven 500 rad whole-body rradaton (Cs 137), and 2-4 h later 107 lve FBL-3 tumor cells were njected nto the rght hnd footpad n 0.05 cc of sterle HBSS. By day 5 tumors were readly palpable and the footpad dameter measured between 2.5 and 3 mm. Also by day 5 ether the rght poplteal lymph node or 0.75 ml of blood from tumorbearng mce could cause progressve tumor growth when njected ntrapertoneally nto a normal mouse that had receved 500 rad. f left untreated, both the footpad tumor and ts metastases grew and eventually klled the anmal by 20 d after the ntal tumor njecton. 5 d after tumor was nduced, when the tumor was clearly measurable n all anmals, mce were randomly assgned to treatment groups and njected ntravenously wth expermental or control cells n 1 cc of sterle HBSS. Each mouse was ear tagged and measured every 2nd or 3rd d n a blnded fashon wthout knowledge of the prevous treatment or of the prevous measurements for that mouse. Spleen Cell Suspenson. Spleens were aseptcally removed, pooled, and crushed gently wth the blunt end of a 10-cc syrnge plunger n HBSS wth 1% fetal calf serum (FCS). The cells were then centrfuged at 500 g for 5 mn. The pellet was resuspended n buffered ammonum chlorde soluton (Natonal nsttutes of Health [NH] Medan Unt) for one mnute at room temperature to lyse red blood cells. The cells were then washed three tmes n sterle HBSS and resuspended n complete medum (CM) before determnaton of cell vablty usng trypan blue. CM was composed of RPM 1640 (Grand sland Bologcal Co., Grand sland, NY) wth 10% heat-nactvated FCS (Grand sland Bologcal Co.), 1/xM sodum pyruvate (Mcrobologcal Assocates, Walkersvlle, MD), 0.1 mm nonessental amno acds (Mcrobologcal Assocates), 0.03% fresh glutamne (NH Meda Unt), s M 2-mercaptoethanol, 100 U/ml penclln, and 100 gg/ml streptomycn. n Vtro Senstzaton (1VS): Condtons for VS have been prevously descrbed (13, 14). n bref, vable responder cells and l0 n rradated stmulator cells were placed n uprght flasks (3013; Costar, Data Packagng, Cambrdge, MA) n 20 ml of CM. Stmulator cells were rradated n a gamma rradator (Cs 137) wth 2000 rad for normal C57BL/6 lymphocytes or 10,000 rad for fresh FBL-3 tumor. Cells were harvested on day 5 and tested n vtro for cytotoxcty, used n experments or placed n L-2. f the cells were njected n vvo, they were frst placed on a 5 ml lymphocyte separaton medum (LSM) gradent (Cedarlane Laboratores,

3 T. J. EBERLEN, M. ROSENSTEN, AND S. A. ROSENBERG 387 Ontaro, Canada) at a concentraton of 107 cells/ml and spun at 1000 g for 20 mn. The nterface was then removed usng a pasteur ppette, and the cells were washed three tmes n HBSS, passed over 100-mesh nylon, counted, and njected. Ths technque resulted n cell vablty >95%. Producton of LF-L-2. Optmal condtons for the producton of L-2 have been prevously descrbed (11). Brefly, exbreeder DBA/2 or BALB/c spleens were aseptcally harvested, mnced, washed, and then ncubated wth 10 pg/ml eoncanavaln A (Con A) (Mles Laboratores, Elkhart, N) for 2 h. The cells were washed three tmes n HBSS and resuspended n CM for 24 h at 37 C and 5% CO2. The resultng culture supernatants were harvested, centrfuged, and poured through 0.45-/~m flters (Mllpore Corp., Bedford, MA). Ths LF-L-2 was >95% Con A free as determned by the absence of mtogenc actvty on fresh lymphoeytes and by measurement of removal of radolabeled Con A (11). Expanson of VS Lymphod Cells. Cells were aseptcally harvested from the VS flasks, centrfuged, and resuspended n fresh CM. Cell vablty was assessed usng trypan blue dye excluson. The senstzed cells were then adjusted to a concentraton of vable cells/ml n an equal volume of CM and LF-L-2. Ths fnal suspenson was placed n 24-well flatbottomed plates (3524; Costar), 2 ml/well. The cells expanded a mnmum of 8.5 tmes the orgnal number n 7 d. f adoptvely transferred, the cells were washed n HBSS three tmes, passed over 100-mesh nylon, counted, and adjusted to the desred concentraton for njecton. All njectons were n 1 ml vol of HBSS. n the experment usng cells expanded multple tmes n L-2, the cells were harvested every 5-7 d, centrfuged at 500g for 5 mn, and readjusted to vable cells/ml n fresh CM:LF- L rradated FBL-3 tumor cells/ml (10,000 rad) were present n the culture from VS untl the fnal 10 d before adoptve transfer. Cells expanded n L-2 were not placed on LSM gradents before adoptve transfer, and always had >95% vablty. Chromum Release Cytotoxcty Assay. An 18-h chromum release assay was used as prevously descrbed (8). Brefly, varyng ratos of effector cells were plated n 96-well round-bottomed 4 51 plates (Lnbro Chemcal Co., Hamden, CT) wth 10 vable Cr-labeled tumor targets per well. The plates were then centrfuged at 80 g for 5 mn, ncubated at 37 for 18 h, and recentrfuged at 400 g for 10 ran, and the supernatants were harvested usng the Ttertek Collectng System (Flow Laboratores, Rockvlle, MD). Fresh FBL-3 was harvested from the asctes of a tumor-bearng mouse, washed, and labeled wth 51Cr. MCA-103 tumor was mnced wth fne scssors, trypsnzed for 7 mn, passed through double-layer 100-mesh nylon, and washed three tmes before 51Cr label. Spontaneous release for these experments was -40%. n prevous studes (13) MCA-103 was shown to be at least as lysable as FBL-3 by an allogenec effector. 18-h assays revealed substantally hgher and more reproducble levels of specfc tumor lyss than were seen n 4-h 5XCr-release assays. e Cytoxcty s expressed as lyrc unts/10 cells. A lytc unt s defned as the number of effector cells that causes 50% lyss of 104 5XCrolabeled target cells. Statstcal Methods. Survval of mce n these experments was computed usng the methods of Peto et al. (17). To compare survval curves, mean survval tme was MST calculated and Student's t-test used to determne P values. No anmals were excluded from statstcal evaluaton. Results Treatment of Dssemnated FBL-3 wth n Vvo mmunzed Lymphod Cells. Adult C57BL/6 female mce were mmunzed to FBL-3 wth one ntramuscular njecton of lve FBL-3. Approxmately 3-4 wk later, ther spleens were harvested and tested n vtro and n vvo. n every experment, these mmune cells showed no n vtro cytotoxcty for FBL-3 tumor n an 18-h 5XCr-release assay (data not shown). When these same cells were adoptvely transferred to mce wth dssemnated footpad tumors, however, they conferred sgnfcant survval beneft when compared wth no treatment or treatment wth smlar numbers of lymphocytes from normal syngenec mce. Fg. 1 s a representatve experment of four experments, each wth a smlar result. The left panel shows the mean footpad tumor sze of each group and the rght panel the

4 388 REGRESSON OF A DSSEMNATED SYNGENEC SOLD TUMOR E 5 u) ~3 k- O 2t ~ ~ ~ "f-' "" -- t~ D-'~5~0" ~" ~ "O t.9 _zeo > ae 40 CURH? No T,,,em~ ore 20 ~ 10l NL Lymphocylml O/ O 2 x 10./nlnltme Lympllocytu O/S Q x 10 7 mmune L y m ~ O/S 10 a mcnchw Lymphocylw 3/4 / TTTT T -,, ;----~ v Fro. t. Footpad tumor sze (left) and survval (rght) of mce wth dssemnated sold FBL-3 lymphoma treated wth n vvo mmunzed lymphocytes. Treatment wth 10 s mmune cells was necessary to completely eradcate footpad tumors and cure anmals. 10 a normal lymphocytes or fewer mmune lymphocytes were not capable of mpactng sgnfcantly on tumor growth or survval. 6O correspondng survval curve. As seen n Fg. 1, 10 s mmune cells gven ntravenously were requred to cure mce. n no experment were mmune cells effectve. Combnng all four experments, 26 of 28 mce gven 10 s mmune cells were cured of tumor, whereas 3 of 17 mce wth normal lymphocytes were cured (P < ). All 20 untreated anmals ded of dssemnated FBL-3 tumor (P < compared to treatment wth mmune cells). Adoptve Transfer of n Vtro Senstzed Lymphod Cells before Expanson n LF-L-2. n vvo mmunzed lymphocytes were resenstzed to rradated FBL-3 tumor n vtro. Smultaneously, nonmmune lymphocytes were co-cultured wth normal C57BL/6 rradated stmulators n vtro. After 5 d n culture, the cells were harvested, washed, placed on an LSM gradent, rewashed three tmes, counted, and adoptvely transferred to mce that had been njected wth tumor n the footpad 5 d earler. No more than vable cells were transferred n any experment. Before transfer, an alquot of the cells was tested for n vtro cytotoxcty. Fg. 2 s a representatve experment (one of three) showng the levels of cytotoxcty attaned after n vtro senstzaton. Only the mmune cells resenstzed to FBL-3 tumor n vtro showed specfc lyss of fresh FBL-3 (91 lyte unts). Nonmmune cells co-cultured wth normal lymphocytes showed nonspecfc lyss, <5 lytc unts, for both fresh FBL-3 and fresh MCA-103 targets. Fg. 3 shows two of the three experments usng treatment wth n vtro senstzed cells. n all three experments, 11 of 14 mce treated wth the mmune cells resenstzed to FBL-3 were cured of tumor, compared wth 0 of 16 mce treated wth nonmmune lymphocytes resenstzed to normal lymphocytes (P < ). Once agan, none of the 18 untreated controls survved (P < ). Adoptve Transfer of Lymphod Cells Expanded n LF-L-2. Cells were harvested from VS flasks, washed, counted, and placed n LF-L-2 at cells/ml. There was a mnmum 8.5-fold expanson of cell number n 7 d. The cells were harvested from the expanson plates, washed three tmes, and adjusted to the approprate cell number n 1 cc of sterle HBSS. Fg. 4 shows the results of one of three smlar experments

5 T. J. EBERLEN, M. ROSENSTEN, AND S. A. ROSENBERG FBL " --~ k=fbl-3 D" (91L.U.) (<1 L.U.) (1.1L.U.) (3.2L.U.) n 60 4O 20 o O.1:1 1:1 10:1 100:1 E/T Fla. 2. n vtro cytotoxcty of mmune cells co-cultured wth FBL-3 tumor for 5 d or nonmmune cells n vtro senstzed to normal C57BL/6 lymphocytes for 5 d. Resenstzaton of mmune cells to FBL-3 tumor conferred specfc cytotoxcty at the hgher effector/target rato (EFT). L.U., lytc unts. demonstratng a hgh degree of specfc lyss of fresh FBL-3 tumor. mmune lymphocytes prevously co-cultured wth FBL-3 tumor and then expanded 8.5-fold n LF- L-2 showed 2,000 lytc unts aganst fresh FBL-3 and <15 lytc unts aganst fresh MCA 103. Nonmmune, n vtro senstzed lymphocytes cultured n L-2 for an equal length of tme showed <5 lytc unts when tested aganst ether fresh tumor target. Nonmmune lymphocytes senstzed n vtro to FBL-3 tumor also demonstrated no lyss of ether tumor both before and after expanson n LF-L-2 (data not shown). Fg. 5 shows the results of the frst experment wth the adoptve transfer of expanded cells n ths dssemnated footpad tumor model. mmune lymphocytes resenstzed to FBL-3 tumor for 5 d and then expanded 8.5-fold n LF-L-2 for 7 d were capable of curng 11 of 12 mce treated wth one ntravenous njecton of ether 5 X 107 or cells (P < ). The only mouse n these groups that ded dd so on day 42 wthout evdence of tumor, and ths anmal receved a lower dose of effector cells ( ). There was no sgnfcant dfference ether n footpad sze (Fg. 5, left) or survval (Fg. 5, rght) between mce recevng 5 X 107 or lymphocytes. Normal lymphoeytes expanded n LF-L-2 for 7 d faled to have any mpact on ether footpad tumor sze or survval. n a repeat experment, the adoptve transfer of cells occurred on day 6 after njecton of tumor n the footpad. Once agan, mmune lymphocytes co-cultured wth FBL-3 tumor for 5 d and then expanded n LF-L-2 for 7 d conferred sgnfcant survval beneft (P < ), wth all but one footpad tumor returnng to normal by day 17. There was one death on day 31 n a mouse wth dssemnated tumor. When these cells receved 2,000 rad just before adoptve transfer (open squares, Fg. 6), the survval beneft was abrogated. When rradated cells were used, no anmals were cured, and the survval tme was the same as n the untreated control group. Thus, t

6 390 REGRESSON OF A DSSEMNATED SYNGENEC SOLD TUMOR O0 E ll N < t- O 0,L z _~ > so m 40 ~' CURES 20 Adoptve ~ 4 x 10 7 left-3 4/ Transfer ~ 4 x 10 7 N~N OS log 8(~ Ll N 4 Q < 3 G ~ o 1 z > f- ~ 4(1 cu~s 20 Adoptve ~ s x to 7 afl-3 4/4 Transfer ~ s x lo 7 NCaNL Or5 NO ~F.A~-NT 0/4 J Fro. 3. Footpad tumor sze (left graphs) and survval (rght graphs) of mce wth dssemnated sold syngenec tumors treated wth cells n vtro senstzed for 5 d n CM. mmune lymphocytes resenstzed to FBL-3 tumor cured 9 of 10 anmals n these two experments. Nonmmune lymphocytes eocuhured to normal C57BL/6 lymphocytes were no more effectve than no treatment. appeared that a cell capable of dvdng n vvo was necessary to medate an ncrease n survval. Table shows the combned result of all three experments performed wth adoptve transfer of cells expanded n L-2 for 1 wk mmune senstzed cells expanded n L-2 cured 93% of all anmals treated cells cured 80% of the anmals, and the one anmal that ded dd so wthout evdence of tumor. A dose of 5 X 106 mmune expanded cells cured three of eght anmals treated. rradaton at 2,000 rad of these same cells totally abolshed ther effectveness n vvo. Smlarly, nonmmune senstzed and expanded cells cured none of the 11 anmals so treated, and all of the 17 untreated control anmals ded. The mmune cells n Fg. 5 were expanded 3,500-fold n LF-L-2 for ~ 1 mo as descrbed n Materals and Methods. These cells mantaned a hgh degree of specfc lyss of fresh FBL-3 tumor (Fg. 7) when tested n an 18-h 5XCr release assay on the day of adoptve transfer (3,571 lytc unts). These same cells conferred sgnfcant survval beneft (Fg. 8) when adoptvely transferred. Fve of nne anmals recevng cells were cured, and three of seven anmals were cured at a dose of 107 transferred cells per mouse (P. 0.01). Nonmmune lyrr, phocytes resenstzed to

7 T. J. EBERLEN, M. ROSENSTEN, AND S. A. ROSENBERG 391 loo FBL _~ afbl-3 Q (2000 L.U.) (13.gL,U,) 4L.U.] N"NL ~r- 14 L.U, 8o o f t a " y '~" E.~...~ - s s,, J.025:1.25:1 2. :1 25:1 FG. 4. n vtro cytotoxcty of cells expanded for 7 d n LF-L-2. mmune cells senstzed to FBL-2 tumor and expanded n L-2 showed ncreased specfc lyss when compared to nonmmune lymphocytes senstzed to normal C57BL/6 lymphocytes and expanded n L-2 for a smlar perod of tme. L.U., lytc unts. E/T ~s E U) 7 6 Expsnlon = ,c~.-- A.'n. 8O (3 z 60 > > n 40 a J-t_ n f CURES 20! -~ ---- S x 107 =FBL-3 (Exp.) 7/7 c~-,-[3 2.5 x 107 k~fbl-3 (Exp.) 4/5 5 x 107 NLaNL (Exp.) 0/5 e---e No Treatnemnt 0/5 [ [ [ Fro. 5. Footpad tumor sze (left) and survval (rght) of mce wth dssemnated sold tumors treated wth cells expanded n L-2 over 7 d. mmune cells senstzed to FBL-3 and expanded for 7 d n L-2 cured all mce treated, and cured 80% when a reduced dose of these cells was gven. Nonmmune cells senstzed and then expanded n L-2 for a smlar perod were no better than the no-treatment control group. normal C57BL/6 stmulators and expanded n LF-L-2 for the same length of tme could not medate the shrnkage of footpad tumors (Fg. 8, left) or mpact on survval (Fg. 8, rght). 6O Dscusson n the experments reported n ths paper, we have demonstrated that mce bearng a palpable local and dssemnated tumor could be cured by the systemc adoptve

8 392 REGRESSON OF A DSSEMNATED SYNGENEC SOLD TUMOR ~S e ~4 7 6 Cl ~3 c~~ 100 Expar~on = 8,6 80 _z 60 > ' cu.es 20 ~ H 5 x 0~ lafjt.-3 xp.),3/4 X 107 lafbl-3 (Exp.)(2000R 014 e---e NO TREATMENT 0/6 1 l ' O 30 FG. 6. Footpad tumor sze (left) and survval (rght) of mce wth dssemnated sold syngenec tumors treated on day 6 wth cells expanded n L-2. mmune cells senstzed to FBL-3 tumor and then expanded n L-2 for 7 d cured three of four mce treated; however, f these same cells were gven 2,000 rad before transfer, ther n vvo effectveness was abolshed. TABLE Footpad Survval Percent survval cures a FBL-3 expanded* 5 X /14 93% 2.5 x /5 80% /4 50% 5 X /8 37.5% 5 X 107 (2,000 rad) 0 0/4 0% NL a NL expanded:t: 5 X /11 0% No treatment 0 0/17 0% * mmune lymphocytes resenstzed to FBL-3 tumor n vtro and expanded n LF-TCGF for 7 d. :~ Nonmmune lymphocytes resenstzed to normal lymphocytes n vtro and then expanded for 7 d n LF-TCGF. transfer of a sngle njecton of n vvo mmunzed cells, n vtro senstzed cells, or senstzed cells expanded >3,000-fold n L-2. These experments are the frst to demonstrate cure of a syngenec sold tumor usng cells expanded for many generatons n L-2. These results also extend our prevous observatons of the treatment of ntrapertoneal FBL-3 tumor. We prevously demonstrated (14) that a combnaton of chemotherapy and adoptve transfer of senstzed cells expanded n L-2 was capable of curng mce wth dssemnated and ntrapertoneal FBL-3 tumor. Smlar results were obtaned by Cheever et al. (18, 19). However, the nterpretaton of our prevous studes (14) and those of others (18, 19) usng the adoptve transfer of cells expanded n L-2 to treat ntrapertoneal FBL-3 tumor were confused by the need for cytoreductve treatment of the tumor-bearng mouse wth cyclophosphamde before cell transfer and by the need to nject the transferred cells ntrapertoneally at the ste of major tumor growth. These problems led us to develop the present model, n whch expanded lymphod cells njected

9 T. J. EBERLEN, M. ROSENSTEN, AND S. A. ROSENBERG t/) ss j~...-- "" 201 s Overall Expanson = 3500 FBL H ofbl-3 O'-"- ] 13571L,U.) 20L.U.) 0.1:1 1:1 10:1 100:1 Fro. 7. n vtro cytotoxcty of cells expanded ~ 1 mo n L-2. mmune cells senstzed to FBL-3 tumor and expanded 3,500-fold n LF-L-2 mantaned specfc lyss of fresh FBL-3 tumor. L.U., lytc unts. EK : =,, bt. lo7,,fal-,~-- 3/7 1 1 dk 5 x 107 NLeNL (3800x) 0/5 2t A. 0 / ' r ~, 10./N..~L ~ z 0/4 10 F- l l L l l s lo 15 2o 25 = 35 lo ~ = Fro. 8. Footpad tumor sze (left) and survval (rght) of mce wth dssemnated sold FBL-3 tumors treated wth cells expanded n L-2 for - 1 too. mmune cells senstzed to FBL-3 tumor and expanded 3,500-fold cured a sgnfcant number of mce at the hgh dose of cells transferred and low dose. No dose of smlarly expanded control cells was capable of cure. 70 ntravenously are used as sole therapy to test the ablty of cells expanded n L-2 to cure mce of syngenec palpable and dssemnated tumor. Ths model has several mportant advantages. The tumor beng treated by adoptve transfer of cells s a palpable sold tumor n the footpad of mce, and the regresson of ths palpable tumor can be easly followed by drect measurement. On the day of

10 394 REGRESSON OF A DSSEMNATED SYNGENEC SOLD TUMOR treatment, tumor s dssemnated both n the blood and n the lymphatcs, and thus treatment effects on local and dstant tumor can be evaluated. Tumor-bearng mce receve treatment wth cells alone, and thus the potentally confusng cytoreductve effects of cyclophosphamde are avoded. Another feature of our new model s that adoptve treatment wth cells s admnstered ntravenously and not nto the ste of the tumor. n prevous studes of the treatment of ntrapertoneal tumor, we (14) and others (18, 19) njected effector cells ntrapertoneally. The possblty exsted, therefore, that adoptvely transferred cells acted manly n local tumor neutralzaton, rather than systemc mmunotherapy. Whole body prerradaton of mce s essental to enable growth of the FBL-3 tumor n the footpad. The effect of ths rradaton on the therapeutc effectveness of the adoptvely transferred lymphocytes s not known, but may be an mportant factor by elmnatng suppressor cells. Berendt and North (5), showed that adoptve transfer of fresh mmune lymphod cells could cause the complete regresson of large establshed tumors only f the tumor bearers were T cell defcent. Furthermore, these authors showed that nfuson of splenc T cells from tumor-bearng donors could nhbt the regresson of establshed tumor n T cell-defcent recpents, confrmng the mportance of host suppressor mechansms n these tumor systems. n ths paper, we have shown that lymph nodes and/or blood from a mouse bearng the FBL-3 tumor 5 d after tumor njecton are capable of transferrng tumor to a normal, rradated, C57BL/6 syngenec recpent. The tumor s thus wdely dssemnated by day 5. Treatment of ths tumor wth n vvo mmunzed cells, on day 5, when t s also clearly palpable, resulted n cure of 93% of all mce treated n four experments. As seen n Fg. 1, however, -10 a cells were requred to cure a mouse. When these n vvo mmunzed cells were resenstzed to FBL-3 n vtro for 5 d, cures could be acheved wth smaller doses of cels (Fg. 3). Ths fndng emphaszes the value of n vtro senstzaton n the actvaton/reactvaton of cells that are necessary for successful adoptve mmunotherapy (20). When approprately senstzed cells were expanded n LF-L-2, they were hghly lytc for fresh FBL-3 tumor n vtro (Fgs. 4 and 7) and capable of curng mce of dssemnated footpad tumors (Fgs. 5 and 6). ndeed, n three experments (as seen n Table ), expanded cells adoptvely transferred to mce wth dssemnated footpad tumors cured mce when as few as cells were transferred. Cells grown n L-2 for almost 1 mo (3,500-fold expanson) were also capable of curng a sgnfcant number of anmals wth dssemnated footpad tumors (Fg. 8). Thus, through the use of L-2, we have been able to expand the therapeutcally effectve cell to suffcent numbers to cure mce. The type of cell effectng the cure of tumor n ths model s not known. t appears that a cell capable of prolferaton n vvo s necessary, and that ths prolferaton takes 7-10 d before a crtcal mass of effector cells s reached to mpact on tumor growth. Although the n vtro senstzed and expanded effector populatons capable of curng anmals n ths model are cytotoxc for fresh FBL-3 tumor n vtro, there s a potental problem n selectng cells for use n adoptve mmunotherapy based solely on n vtro reactvty. The prmary effector cells n vvo n ths model may not be the cytotoxc cell measured n our n vtro assays. Fernandez-Cruz et al. (6) showed that

11 T. J. EBERLEN, M. ROSENSTEN, AND S. A. ROSENBERG 395 the subset of T cells most effectve n eradcatng rat sold tumors n vvo were noncytotoxc n an n vtro 51Cr release assay. Supportng the hypothess that a helper cell s the prmary effector cell n medatng tssue rejecton s the work of Loveland et al. (20, 21), who showed that skn and tumor allograft rejecton n vvo was dependent on the presence of Lyt-1 cells. Greenberg et al. (22) reported smlar fndngs used n vvo mmunzed cells depleted of Lyt-2 cells to cure mce of dssemnated FBL-3 tumor. Smlar fndngs were obtaned by Fernandez-Cruz (23) n a syngenec rat sold tumor model. Regardless of whch cell s effectve n our tumor model, we were able to ncrease the number of cells avalable for adoptve transfer usng L-2. The demonstraton that ntravenously njected lymphod cells expanded n L-2 are capable of medatng local and dssemnated tumor regresson has mportant mplcatons for the adoptve mmunotherapy of tumors. The abnormal traffc patterns seen when cells expanded n L-2 are renjected nto mce and humans (24) have led to concern that these expanded cells would not be functonal when njected n vvo. Our prevous demonstraton that ntravenous njecton of cells expanded n L-2 was capable of acceleratng the rejecton of allogenec skn grafts (12), and the present demonstraton that syngenec tumors could be made to regress followng ntravenous njecton of expanded senstzed cells ndcate that cells expanded n L-2 can dstrbute approprately and medate mmunologc tssue destructon followng ntravenous njecton. Our current efforts are drected at senstzng autologous lymphod cells to human tumors n the hope that expanson of these cells wll be useful n the adoptve mmunotherapy of human tumors. Summary We have studed the ablty of mmunzed lymphod cells expanded n L-2 to medate the cure of mce wth localzed and dssemnated syngenec lymphoma. Mce receved 500 rad total-body rradaton before njecton of tumor nto the footpad. Mce were treated 5 d later when a palpable local tumor and dssemnated metastases were present. ntravenous njecton of n vvo mmune lymphocytes cured 93% of all mce, sgnfcantly better than any control group (P < ). mmune cells, secondarly senstzed to the FBL-3 tumor n vtro, also conferred sgnfcant survval beneft (P < ) when njected ntravenously, curng 79% of the anmals treated. When these n vtro senstzed cells were expanded n L-2, 8-10-fold over 7 d, 93% of the anmals thus treated were cured, (P < ). When these cells were grown for multple generatons n L-2 they retaned ther ablty to cure mce (56% cured, P < 0.01). Ths s the frst demonstraton that ntravenous njecton of senstzed cells grown n long term culture n L-2 s capable of curng mce of establshed local and dssemnated syngenec tumor. Receved for publcaton 12 Aprl References 1. Rosenberg, S. A., and W. Terry Passve mmunotherapy of cancer n anmals and man. Adv. Cancer Res. 25:323.

12 396 REGRESSON OF A DSSEMNATED SYNGENEC: SOLD TUMOR 2. Delorme, E. J., and P. Alexander Treatment of prmary fbrosarcoma n the rat wth mmune lymphocytes. Lancet. : Alexander, P., E. J. Delorme, and J. G. Hall. t966. The effect of lymphod cells from the lymph of specfcally mmunzed sheep on the growth of prmary sarcomata n rats. Lancet. : Borberg, H., H. F. Oettgen, K. Choudry, and E. J. Beatte, Jr nhbton of establshed transplants of chemcally nduced sarcoma n syngenec mce by lymphocytes from mmunzed donors. nt. J. Cancer. 10: Berendt, M. J., and R. J. North T-cell-medated suppresson of ant-tumor mmunty. An explanaton for progressve growth of an mmunogenc tumor. J. Exp. Med. 151: Fernandez-Cruz, E., B. A. Woda, and J. D. Feldman Elmnaton of syngenec sarcomas n rats by a subset of T-lymphocytes.J. Exp. Med. 152: Rosenberg, S. A., P. J. Spess, and S. Schwarz n vtro growth of murne T-cells.. Producton of factors necessary for T-cell growth.j. mmunol. 121: Rosenberg, S. A., S. Schwarz, and P.J. Spess n vtro growth of murne T-cells.. Growth of n vtro senstzed cells cytotoxe for alloantgens.j. mmunol. 121: Glls, S., and K. A. Smth Long-term culture of tumor-specfc cytotoxc T-cells. Nature ( Lond. ). 268: Rosenberg, S. A., P. J. Spess, and S. Schwarz n vtro growth of murne T cells. V. Use of T cell growth factor (TCGF) to clone lymphod cells. Cell. mmunol. 54: Spess, P. J. and S. A. Rosenberg A smplfed method for the producton of murne T cell growth factor free of lectn. J. mmunol. Methods. 42: Rosensten, M., T. J. Eberlen, M. M. Kemeny, P. H. Sugarbaker and S. A. Rosenberg n n vtro growth of murne T-cells. V. Accelerated skn graft rejecton caused by adoptvely transferred cells expanded n T-cell growth factor. J. mmunol. 127: Eberlen, T. J., M. Rosensten, P. J. Spess, and S. A. Rosenberg The generaton of long term T-lymphod cell lnes wth specfc cytotoxc reactvty for a syngenec murne lymphoma. J. Natl. Cancer nst. n press. 14. Eberlen, T. J., M. Rosensten, P.J. Spess, R. Wesley, and S. A. Rosenherg Adoptve chemommunotherapy of a syngenec murne lymphoma usng long term lymphod cell lnes expanded n T-cell growth factor. Cancer mmunol. mmunother. n press. 15. Glynn, J. P., J. L. McCoy, and A. Fefer Cross resstance to the transplantaton of syngenec Frend, Moloney and Rauscher vrus-nduced tumors. Cancer Res. 28: Parker, G. A., and S. A. Rosenberg Serologc dentfcaton of multple tumorassocated antgens on murne sarcoma. J. Natl. Cancer nst. 58: Peto, R., M. Pke, P. Armtage, N. Breslow, D. Cox, S. Howard, N. Mantel, K. McPherson, J. Peto, and P. Smth Desgn and analyss of randomzed clncal trals requrng prolonged observaton of each patent.. Analyss and examples. Brt. J. Cancer. 35: Cheever, M. A., P. D. Greenberg, and A. Fefer Specfc adoptve therapy of establshed leukema wth syngenec lymphocytes sequentally mmunzed n vvo and n vtro and nonspecfcally expanded by culture wth nterleukn 2. J. mmunol. 126: Cheever, M. A., P. D. Greenberg, and A. Fefer Tumor neutralzaton, mmunotherapy and chemommunotherapy of a Frend leukema wth cells secondarly senstzed n vtro.. Comparson of cells cultured wth and wthout tumor to noncultured mmune cels..]. mmunol. 121: Loveland, B. E., P. M. Hogarth, R. Ceredg, and. F. C. McKenze Cells medatng graft rejecton n the mouse.. Lyt-1 cells medate skn graft rejecton.j. Exp. Med. 153: Loveland, B. E., and. F. C. McKenze Cells medatng graft rejecton n the mouse.. The Ly phenotypes of cells producng tumor allograft rejecton. Transplantaton (Baltmore). 33: Greenberg, P. D., M. A. Cheever, and A. Fefer, Eradcaton of dssemnated murne

13 T. J. EBERLEN, M. ROSENSTEN, AND S. A. ROSENBERG 397 leukema by chemommunotherapy wth cyclophosphamde and adoptvely transferred mmune syngenec Lyt-l+2 - lymphoeytes.j. Exp. Med. 154: Fernandez-Cruz, E., S. C. Glman, and J. D. Feldman mmunotherapy of a chemcally-nduced sarcoma n rats: characterzaton of the effector T-cell subset and nature of suppresson. J. mmunol. 128: Lotze, M. T., B. R. Lne, D. J. Mathsen, and S. A. Rosenberg The n vvo dstrbuton of autologous human and murne lymphod cells grown n T-cell growth factor (TCGF): mplcatons for the adoptve mmunotherapy of tumors. J. mmunol. 125:1487.