[CANCER RESEARCH 55, 3369-3373, August 1, 1995]

In Vivo Antitumor Activity of T Cells Redirected with Chimeric Antibody/T-Cell

Receptor GenesP. Hwu,1 J. C. Yang, R. Cowherd, J. Treisman, G. E. Shafer, Z. Eshhar, and S. A. Rosenberg

Surgery Branch. National Cancer Institute. Bethesda, Maryland 20892 ¡P.H., J. C. Y., R. C., J. T., G. E. S., S. A. R.¡;and Department of Chemical Immunology, WeidmannInstitute of Science. Rehomt 76100. Israel ¡Z.E.¡

ABSTRACT

In an effort to broaden the applicability of adoptive cellular immuno-therapy toward nonmelanoma cancers, we have designed chinici ir ¡iiiti-body/T-cell receptor genes composed of the variable domains from niAhsjoined to T-cell receptor-signaling chains. We have demonstrated that Tcells retrovirally transduced with these genes can recognize antibody-defined antigens and that this recognition leads to T-cell activation,

specific lysis, and cytokine release.In this study, we have examined the in vivo activity of murine T cells

transduced with a chimeric receptor gene (MOv-y) derived from the m \l>MO\ IS, which binds to a folate-binding protein overexpressed on most

human ovarian adenocarcinomas. Nude mice that were given i.p. implantsof human ovarian cancer (IGROV) cells were treated 3 days later with i.p.murine tumor-infiltrating lymphocytes (TIL) derived from an unrelatedtumor. Mice treated with MOv-y-transduced TIL (MOv-TIL) had signif

icantly increased survival compared to mice treated with saline only,nontransduced TIL, or TIL transduced with a control anti-trinitrophenylchimeric receptor gene (TNP-TIL). In another model, C57BL/6 mice weregiven i.v. injections of a syngeneic methylcholanthrene-induced sarcomatransduced with the folate-binding protein i / /(/' i gene. Three days later,

mice were treated i.v. with various transduced murine TIL (derived froman unrelated tumor), followed by low-dose systemic interleukin 2. Eleven

days after tumor injection, mice were sacrificed, and lung métastaseswerecounted. In multiple experiments, mice receiving MOv-TIL had signifi

cantly fewer lung métastasesthan did mice treated with interleukin 2alone, nontransduced TIL, or TNP-TIL.

These studies indicate that T cells can be gene modified to react in vivoagainst tumor antigens, defined by mAbs. This approach is potentiallyapplicable to a number of neoplastic and infectious diseases and may allowadoptive immunotherapy against types of cancer not previously amenableto cellular immunotherapy.

INTRODUCTION

T-cell based therapies utilizing IL-22 and tumor-reactive T cells

such as TILs have been developed for patients with metastatic melanoma, resulting in significant regression of tumor in some patients(1, 2). The nature of T-cell recognition of melanoma is being elucidated, with the cloning and characterization of several melanoma-associated antigens recognized by T cells (3-8). However, applying

these principles to other, more common types of cancer such as breastcancer and colon cancer has remained elusive, largely due to difficultyin obtaining T cells with specific reactivity against these cancers.

mAbs have been generated that recognize tumor-associated anti

gens common to certain cancers. Therapy with mAbs alone haslargely been ineffective due to weak effector responses, inadequatetissue penetration, or the development of human anti-mouse antibody

responses (9). To combine the tumor recognition capabilities of antibodies with the potent antitumor effector abilities of T cells, we have

Received 1/23/95; accepted 5/30/95.The costs of publication of this article were defrayed in part by Ihe payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' To whom requests for reprints should be addressed, at National Cancer Institute,

Surgery Branch, Building 10, Room 2B42, 9000 Rockville Pike, Bethesda, MD 20892.~The abbreviations used are: IL-2, interleukin 2; TIL, tumor-infiltrating lymphocyte:

TNP, Irinitrophenyl; FBP, folate-binding protein; FACS, fluorescence-activated cell sorting;mIFN, murine IFN: TCR, T-cell receptor.

designed chimeric receptor genes encoding the variable domain frommAbs joined to T-cell-signaling chains. Previously, we demonstrated

that retroviral transduction of T cells with these chimeric receptorgenes could enable T cells to recognize, lyse, and specifically secretecytokines upon contact with cells expressing the appropriate,antibody-defined antigen in vitro (10, 11).

In this report, we have examined the ability of murine T cellsexpressing these chimeric antibody/T-cell receptors to function in vivoin tumor-bearing mice. The ability of these transduced T cells to

successfully treat established tumors in vivo has significant implications in the generalization of this approach to the treatment of avariety of human cancers and infectious diseases.

MATERIALS AND METHODS

Construction of Chimeric Receptor (¿cues. Chimeric receptor genescomposed of single chain variable regions from mAbs joined to the Fc receptory chain, which is capable of mediating TCR signal transduction ( 12-16). were

constructed as described previously (10, 11). Chimeric receptors derived fromMOvlS (17, 18), a mAb that binds a M, 38,(XK)FBP highly expressed on mostovarian adenocarcinomas, and Sp6 (19, 20), an anti-2,4,6 TNP mAb, wereengineered as described (11) (M0v--y and Sp--y receptors, respectively).

Retroviral Vectors. The MOv-y or Sp-y chimeric receptor genes were

cloned into the LXSN or G1EN (11, 21, 22) retroviral backbones under thetranscriptional control of the long terminal repeat from Moloney murineleukemia virus. The retroviral constructs also contained the neomycin phos-photransferase gene (NeoR) as a selectable marker.

The gene encoding FBP was obtained from L. Coney (Apollon, Malvern,PA) and cloned into the LXSN retroviral backbone. The retroviral constructswere then Iransfected with the use of CaPO4 into the PA317 amphotropicpackaging cell line as described previously (11, 23).

Tumor Transduction and Cell Culture. Tumor cell lines were cultured inRPMI 1640 with 10% heat-inactivated PCS and glutamine (all from Biofluids,Rockville. MD). 24JK tumor cells, a clone from the 3-methylcholanthrene-

induced poorly immunogenic MCA 102 murine sarcoma (24, 25), were transduced with the FBP gene by incubation in retroviral supernatant in thepresence of 8 pig/ml polybrene (Aldrich Chemical Co., Milwaukee, Wisconsin)to yield the 24JK-FBP tumor line. Media was replaced with fresh retroviralsupernatant and polybrene every 12 h for 3 days. Seventy-two h after the final

supernatant change, tumor cells were selected in 400 /j.g/ml of the neomycinanalogue G418 (GIBCO, Grand Island, NY). After G418 selection, successfultransduction was demonstrated by FACS analysis of tumor cells with MOvlS

mAb.Lymphocyte Transduction and Cell Culture. Murine TILs, derived from

the diphenylhydrazine-induced MC38 murine colon adenocarcinoma, weregrown in IL-2 as described (26). For retroviral transduction with MO\--y andSp-y chimeric receptor genes, (to generate MOv-TIL and TNP-TIL, respectively), antigen-stimulated TIL were pelleted and resuspended at 3 X IO5

TIL/ml in retroviral supernatant containing 30 lU/ml human recombinant IL-2

and 20 fig/ml protamine sulfate (Eli Lilly & Co., Indianapolis, IN). Media waspartially exchanged with fresh retroviral supernatant containing IL-2 andprotamine sulfate every 12 h for 1-2 additional exposures. Forty-eight h after

the final supernatant change, TILs were selected in 0.3 mg/ml G418 for 5 days.This was followed by 1 week of expansion without G418, and then another5-day selection in 0.3 to 1 mg/ml G418. After G418 selection, successful

transduction was confirmed by Northern analysis of total RNA as described(27).

mIFN-y ELISA. TIL (5 X IO5) and stimulator (5 x IO5) cells werecocultured for 24 h at 37°Cin a final volume of 1 ml RPMI with 10% PCS and

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30 IU/ml of IL-2. Supernatants were then aspirated, centrifugea at 2000 rpm toremove cells, decanted, and frozen at -70°C. Thawed aliquots were tested by

ELISA for murine IFN-y. The ELISA used a solid-phase rat IgG2A mAbspecific for murine IFN-y (Life Technologies, Gaithersburg, MD). Afteraddition of either sample or recombinant IFN-y standard, a biotinylated ratIgGl mAb specific for IFN-y (PharMingen, San Diego, CA) was used,followed by avidin-peroxidase. Color reaction was performed with the additionof H,O2 and ABTS substrate [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic

acid); Sigma Chemical Co., St. Louis, MO). The plates were then read at Ams.Mice. C57BL/6 mice were obtained from Charles River Breeding Labora

tories (Raleigh, NC) and the Frederick Cancer Research Facility (Frederick,MD) and were used at 8-16 weeks of age. Athymic nude mice were obtained

from the Frederick Cancer Research Facility, maintained in laminar flowhousing, and used at 6-12 weeks of age.

Pulmonary Metastasis Tumor Therapy Model. C57BL/6 mice received500 cGy total-body irradiation (to minimize any host antitumor immuneresponse), followed by i.v. injection of 5 X IO5 to 1 X 10" 24JK or 24JK-FBPtumor cells. On day 3, mice were treated i.v. with 2-3 x IO7 transduced or

nontransduced TIL cells derived from the MC38 tumor, followed by 30,000 to60,000 1U IL-2 i.p. 3 times a day for 9 doses. Eleven to 16 days after initial

tumor injection, mice were ear tagged and randomized, and the lungs wereremoved; the number of pulmonary métastaseswas evaluated in a coded,blinded fashion as described previously (28). Lungs with >250 métastaseswerescored as a250 because this was the largest number that could be accuratelycounted. Numbers presented are the mean numbers of pulmonary métastases ±SE. The significance of differences between groups was determined withthe Wilcoxon rank Sums test. All P values are two-tailed.

Intraperitoneal Tumor Therapy Model. IGROV-1 human ovarian cancer

cells (29, 30) were adapted in vivo by serial i.p. passages in nude mice until theline consistently grew as ascites. For the i.p. tumor model. 2.5 X IO6 IGROV-1

fresh ascites cells were washed and injected i.p. into nude mice. Three dayslater, the peritoneal tumor burden was evaluated in sample mice, and theremainder was treated with a single i.p. injection of 1-3 X IO7 nontransduced

or transduced MC38 TIL cells. Mice were then ear tagged and randomized toavoid cage effects and followed for survival.

RESULTS

Gene Transfer of FBP Antigen into Nonimmunogenic MurineSarcoma. The nonimmunogenic murine fibrosarcoma 24JK was ret-

rovirally transduced with the gene encoding FBP, the antigen recognized by MOvlS. After selection with the neomycin analogue G418,FBP-transduced 24JK tumor (24JK-FBP) displayed high levelsof FBP, as did the human ovarian carcinoma IGROV-1, as measured

by FACS analysis with MOvlS (Fig. 1).

24JK

Fig. 1. FACS analysis with mAb MOvlS to determine expression of FBP on varioustumor lines. IGROV-1, human ovarian carcinoma. 24JK, clone of the poorly immunogenicmethylcholanthrene induced murine sarcoma MCA-102. 24JK cells were retrovirallytransduced with a vector containing the FBP gene to generate the 24JK-FBP cell line. 888

MEL, human melanoma cell line. Open graph, control antibody; shaded graph, MOvlSantibody; ordinate, relative number of cells; abscissa, fluorescence intensity.

In Vitro Function of Murine TIL Transduced with Chimi-rieReceptor Genes. Murine TIL derived from the MC38 colon adeno-

carcinoma (38 TIL) were transduced with chimeric receptor genesderived from either the anti-ovarian cancer mAb MOvlS (MOv-y) orthe anti-TNP mAb Sp6 (Sp-y; Ref. 11), and selected in G418. Toassess in vitro activity, transduced, G418-selected TILs were cocul-tured with tumor lines for 16-24 h. Supernatants were then harvestedand analyzed for mIFN-y by ELISA. All TIL cultures produced largeamounts of mIFN-y when cocultured with MC38 tumor cells (theirnative antigen) or in anti-CD3-coated plates. When cocultured withIGROV-1 or 24JK.-FBP tumor cells, both expressing large amounts ofFBP, mIFN-y production by MOv-y-transduced TIL (MOv-TIL) increased by 54- and 14-fold, respectively, compared to MOv-TILalone. In contrast, mIFN-y production by nontransduced TIL andTIL transduced with the anti-TNP Sp-y receptor (TNP-TIL) increasedby only 2-4-fold upon coculture with the FBP-expressing cell lines,and was not different compared to coculture with the FBP-nonex-

pressing cell lines. None of the TIL cultures produced substantialamounts of mIFN-y upon coculture with nontransduced 24JK cells or

888 human melanoma cells (Table 1). These data indicate that theMOv-y receptor gene can confer to murine TIL the capability tospecifically recognize FBP-expressing tumor cells.

Treatment of Pulmonary Métastases.To determine whetherMOv-TIL had antitumor activity in vivo, C57BL/6 mice were giveninjections via the tail vein of 1 X 10ft 24JK tumor cells that were

either nontransduced or transduced with the FBP gene. Three dayslater, mice were treated with 2.7 X IO7 TIL, followed by 60,000 ID

IL-2 every 8 h for 9 doses. Eleven days after the initial injection of

tumor cells, mice were sacrificed, and lung métastaseswere counted.Only treatment with MOv-TIL in combination with IL-2 resulted in a

significant reduction in lung métastases(P2 < 0.0004 compared to allother treatment groups), whereas treatment with IL-2 alone or non-transduced TIL in combination with IL-2 did not significantly reducethe number of 24JK-FBP pulmonary métastases(Table 2 and Fig. 2).MOv-TIL did not reduce the number of nontransduced 24JK tumorcells (Table 2) thus demonstrating their specificity for FBP-expressing

tumors. These findings were corroborated in 2 replicate experiments.Treatment of Human Ovarian Cancer Cells in Nude Mice. To

assess whether MOv-TIL had significant in vivo activity againsthuman ovarian carcinoma cells, 2.5 X IO6 IGROV-1 cells from fresh

ascites were implanted i.p. in nude mice. Three days later, mice weretreated with a single i.p. injection of TIL and then followed forsurvival. Histopathological evaluation of sample mice at the time oftreatment revealed that significant amounts of disease were presentand invading structures within the murine peritoneal cavity 3 daysafter tumor injection (Fig. 3). Mice treated with MOv-TIL had significantly increased survival (median survival = 90 days; P2 < 0.002)

compared to mice treated with saline only, nontransduced TIL, orTNP-TIL (median survivals = 31, 37, and 31 days, respectively;

Fig. 4). This study was repeated with similar results.

DISCUSSION

The ability to alter T-cell specificity with the use of chimeric

receptor genes has the potential to broaden the applicability of adoptive cellular immunotherapy to include the treatment of commoncancers such as breast cancer and colon cancer, as well as viraldiseases such as hepatitis B or HIV. In addition, by eliminating theneed to isolate naturally occurring T cells with a particular antigenspecificity, T cells can be selected for a certain phenotype, function,or cytokine profile, regardless of the specificity of the native TCR.Indeed, specific patterns of cytokine production by T cells, such as

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Table 1 IFN-y release by MOv-y-lransduced TIL!'

Stimulator (pg mIFN--y/ml/5 X IO5 cells/16 h)

Responder None 38 Tumor 24JK 24JK-FBP IGROV 888 MEL 2C11*

None38TILNV38

TNP-T1L38MOv-TIL17345338729926147,50034,31541,19547712209468911891708104543242611642100615,9994146887216121483>50,000>50,000>50,000

" In each ml, 5 X IO5 TIL were cocultured for 16 h at 37°Cwith nothing, or 5 X IO5

target cells (e.g., IGROV, 888 MEL), or 2C11 (anli-CD3) antibody.2C11 used at 4 ng/ml, coated with HCO3 buffer, overnight at 4°C.

Table 2 Treatment of pulmonary métastaseswith MOv-TIL

Tumor24JK-FBP24JKTreatmentHBSS

IL-2*NormalTIL' + IL-2

MOv TIL' +IL-2HBSS

IL-2*MOvTIL' + IL-2n8

101075

95Mean

no. lung metastases/mouse"229232195

13"240

221223SEM18.6

11.016.3

3.09.611.07.6

" Mice were sacrificed on day 11 after tumor injection, and lung-metastases were

counted in a blinded fashion. Métastaseswhich were too numerous to count are arbitrarilydesignated as 2250.

' IL-2 was given beginning on day 3 after tumor injection at 6U.OOUIU i.p. 3 times a

day for 9 doses.' TIL (2.7 X 10 , either unmodified or transduced with the MOv-y chimeric receptor)

were given once on day 3 after tumor injection and followed by systemic IL-2 given as

described above.' Significantly less compared to other groups; P < 0.0004.

TH1 versus TH2 cells, have been shown to dictate different immu-nological responses and therapeutic outcomes (31-35).

This study demonstrates that T cells transduced with chimericreceptor genes are active in vivo against tumor cells bearing thereceptor-defined antigen. Previous studies using nontransduced

murine and human TIL (36, 37) have correlated specific cytokineproduction in vitro with function in vivo against tumor cells bearingnative tumor-associated antigens. The present results, using T cells

expressing chimeric receptors, also demonstrate that T cells that aretherapeutically effective in vivo specifically secrete cytokines in vitro.This suggests that chimeric antibody/TCRs may function similarly tonative TCRs with regard to induction of cytokine secretion and in vivoactivity. Besides specific cytokine release, MOv-y-transduced T-cellshave been shown to specifically lyse FBP-expressing target cells in vitro

(11). Further studies are needed to assess the relative contributions ofcytokine release and lysis to the observed in vivo antitumor activity.

Because antibody-based recognition of tumor is dependent on expression of tumor-associated antigens, one potential escape mechanism for tumor cells is the down-regulation of antigen expression. In

the present study, i.p. injection of IGROV tumor cells into nude mice,followed by i.p. therapy with MOv-TIL, resulted in a significantincrease in survival. Although survival was enhanced 3-fold, all mice

eventually died from tumor ascites. FACS analysis of these tumorcells showed continued presence of FBP expression, unchanged fromascites in control mice (data not shown). This suggests that antigendown-regulation was not the mechanism of escape in this particular

model. Complete tumor eradication may require repeated treatments,combinations of i.p. and i.v. therapy, or combinations with othertreatment approaches. Tumor therapy using a variety of chimericreceptors targeting different antigens may also be necessary shouldantigen down-regulation or in vivo immunoselection of antigen-neg

ative cells become evident.An i.p. tumor model is particularly appropriate for ovarian cancer

because the most common and earliest mode of dissemination of thisdisease in cancer patients is by exfoliation of cells that implant along

the surfaces of the peritoneal cavity (38). Alternative tumor modelsmay be more appropriate when studying chimeric receptors targetedagainst other types of cancers.

Although MOv-TIL specifically secreted IFN--y upon coculturewith FBP-expressing cells, more IFN--y was released upon

coculture with the human ovarian cancer cell line (IGROV) compared to the FBP-transduced murine tumor cell line (24JK FBP),

despite the fact that the 24JK FBP line expressed more FBP byFACS analysis. The reason for this difference is not clear. Onepossibility is that free FBP is shed by the high-expressing 24JKFBP line, thereby inhibiting reactivity by the MOv-TIL, as we have

demonstrated previously that free antigen can block reactivity inthis system (11).

This study has utilized primary, differentiated lymphocytes as effector cells. However, with the ability to dictate the specificity of anygiven lymphocyte by retroviral transduction with a particular chimericreceptor gene, other hematopoietic cells could potentially be used aseffectors. For example, retrovirally transduced bone marrow stemcells could be used, thereby providing a constant supply of differentiated cells expressing the chimeric receptor. In addition, by thismethod, not only would lymphocytes contain the gene, but other cellssuch as monocytes and granulocytes might also express the chimericreceptor, thereby providing a continuous supply of multiple types ofeffector cells directed against the tumor. Because TCR and Fc receptor-signaling chains share common activation motifs (12-14, 16), a

variety of effector cells, expressing either Fc receptors or TCRs, maybe capable of functioning via the same chimeric receptor gene. Thechimeric receptor genes we have used for this study have utilized the Fcreceptor y chain for signal transduction. We are currently evaluatingreceptors that utilize the TCR £chain for signal transduction, althoughpreliminary results suggest that the ¥c-ychain constructs are equivalent

or superior to the f chain constructs in our system. This may be due todifferences in surface expression between the two types of receptors.

For this study, we used a murine TIL line that we could readilytransduce. However, we have found that some primary T-cell lines are

Fig. 2. Appearance of lungs at 11 days after injection of 24JK-FBP cells. On day 3 aftertumor injection, mice began therapy with either IL-2 (top left), unmodified TIL + IL-2(top right), or MOv-y transduced TIL + IL-2 (bottom) as described in "Materials andMethods." Lungs were harvested after injection of India ink into trachea. Lungs werebleached with Fekete's solution to produce white métastaseson a black background. Asubstantial reduction in métastaseswas seen in mice receiving MOv--y-transducedTIL + IL-2, compared to the other groups.

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ANTITUMOR ACTIVITY OF RECEPTOR-TRANSDUCED T CELLS

Fig. 3. Histopathological evaluation of peritoneal cavity 3 days after i.p. injection of 2.5 X IO6

human ovarian cancer IGROVOvarian cancer cells can he se

/»*.

mRtf^m^fciyxj,.***.^.V

x;•?r*.':^ï*..^-»-i ~-'•\

Vtx^ •_.•'- ••-

m&!gg&%" -:c-.•Ì&:éeg£^':?Ì'*"Ö^ÄÄiVr-, >•

C",/

.

a

100%

80%

60%

40%

20%

INVTII,

10 20 30 40 50 60 70 80 90 100 110 120

Days after I.P. IGROV

Fig. 4. Survival of nude mice after i.p. injection with human ovarian cancer (IGROV)cells. On day 3 after tumor injection (see Fig. 3 lor histopathological evaluation on day 3),mice were treated with HBSS, unmodified murine TIL, or TIL transduced with cither theMOv-y receptor or the control Sp-v receptor (MOv-TIL and TNP-TIL. respectively).Mice treated with MOv-TIL demonstrated a significant increase in survival compared to

the other groups.

more difficult to genetically modify. Therefore, current efforts arefocused on enhancing transduction efficiencies and gene expression inother primary T-cell lines, as well as bone marrow stem cells, to

maximize the applicability of this technology. For example, we areintroducing the Mov-y receptor into OKT3-stimulated peripheral

blood lymphocytes because these cells are readily accessible andeasily grown from all patients and would thus be suitable effector cellsfor use in clinical trials for the treatment of patients with advancedovarian carcinoma.

ACKNOWLEDGMENTS

We thank L. Coney, S. Canevari, D. Perry-Lalley, A. Mixon, D. Jones, and

P. Spiess for helpful reagents and discussions.

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1995;55:3369-3373. Cancer Res   P. Hwu, J. C. Yang, R. Cowherd, et al.   Antibody/T-Cell Receptor Genes

Antitumor Activity of T Cells Redirected with ChimericIn Vivo

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