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Cancer Immunol Immunother (1991) 32:342-352 034070049100012U

ancer mmunology mmunotherapy

© Springer-Verlag 1991

Retrovirus-mediated gene transfer into CD4 + and CD8 + human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells Shoshana Moreckil, Evelyn Karson 2, Kenneth Cornetta2, Attan Kasidl, Paul Aebersoldl, R. Michael Blaese 3, W. French Anderson 2, and Steven A. Rosenbergl 1 Surgery and 2 Metabolism Branches, National Cancer Institute, and 3 Laboratory of Molecular Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20 892, USA Received 17 May 1990/Accepted 30 August 1990

Summary. Studies were undertaken to test the susceptibility of individual T cell subpopulations to retroviral-mediated gene transduction. Gene transfer into human tumor-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) was carried out by transduction with an amphotropic murine retroviral vector (LNL6 or N2) containing the bacterial neo R gene. The presence of the neo R gene in the TIL population was demonstrated by Southern Not analysis, detection of the enzymatic activity of the gene product and by the ability of transduced TIL to proliferate in high concentrations of G418, a neomycin analog that is toxic to eukaryotic cells. The presence of the n e o R gene in TIL did not alter their proliferation or interleukin-2 dependence compared to nontransduced TIL. The differential susceptibility of CD4 ÷ and CD8 ÷ lymphoid cells to the retro-virus-mediated gene transfer was then tested. Transduction of heterogeneous TIL cultures containing both CD4 + and CD8 ÷ cells resulted in gene insertion into both T cell subsets with no preferential transduction frequency into either CD4+ or CD8 ÷ cells. In other experiments highly purified CD4 ÷ and CD8 ÷ T cell subpopulations from either TIL or PBMC could be successfully transduced with the n e ß gene as demonstrated by Southem blot analysis and detection of the gene product neophosphotransferase activity. No such activity or vector DNA could be detected in controls of nontransduced cells. In these highly purified cell subsets the distinctive T cell phenotypic markers were continually expressed after transduction, G418 selection and long-term growth. Clinical trials have begun in patients with advanced cancer using heterogeneous populations of CD4 ÷ and CD8 ÷ gene-modified TIL.

* Current address: Bone Marrow Transplantation, Hadassah University Hospital, 91120 Jerusalem, Israel Offprint requests to: S. A. Rosenberg, Chief of Surgery, NCI, National

Institutes of Health, Bldg. 10, Room 2B42, Bethesda, MD 20892, USA

Introduction Lymphoid cells are being increasingly considered as suitable cellular vehicles for the introduction of exogenous genes into humans. Tumor-infiltrating lymphocytes (TIL) derived from fresh solid tumors can be expanded to large numbers in culture when incubated with interleukin-2 (IL-2) [1, 10, 21, 31]. TIL have been studied extensively because of their potent anti-tumor properties in murine models [25, 28] and in human patients with advanced metastatic disease [17, 26]. Traffic studies with indium111-1abeled human TIL have shown that they can recirculate and preferentially localize at tumor sites several days after infusion [8]. The radiolabeled cells, however, are not useful for long-term tracking owing to the short half-life of the radiolabel and the tendency of the cells to release 111In spontaneously. In an attempt to study the long-term survival and distribution of TIL following adoptive transfer into humans, techniques were developed to introduce into TIL the bacterial neoR gene as a permanent "marker" using the retroviral vector N2 [7], an amphotropic construct based on Moloney murine leukemia virus (MoMuLV). Initially, six TIL cultures that had been transduced with N2 and selected in G418 (a neomycin analog that is toxic to eukaryotic cells) maintained their pattem of cytotoxicity, DNA rearrangement of [3 and 7 chains of the T cell receptor and cytokinemRNA expression [14] (Culver et al.) unpublished results. Some transduced TIL cultures, however, showed minor shifts in the relative numbers of CD4 ÷ and CD8 ÷ cells compared to cultures of their nontransduced counterparts. Because amphotropically packaged N2 could not be maintained free of recombinant replication-competent retrovirus, we elected to use a related vector, LNL6 [2], in which several modifications of N2 were designed to reduce the possibility of recombination events occuring, thereby preventing the production of helper virus. In the experiments reported here we extended our previous work to study TIL transduction using the retroviral vector LNL6, which is being used for marking TIL in our clinical trials in patients with advanced cancer [26@ Since TIL cultures

343

orten contain a mixed population of CD4 + and CD8 +cells [1, 10, 21, 31], it was important to test whether these two T cell subsets have differential susceptibility to the retrovirus-mediated gene transfer. We describe here studies in which heterogeneous TIL populations or highly purified CD4 + and CD8+ T cell subsets from TIL and PBMC were transduced with the LNL6 retroviral vector. Evidence for the presence of the transferred gene in both cell subsets has been demonstrated.

Materials and methods Cell source and culture conditions. Peripheral blood mononuclear cells (PBMC) were separated from fresh heparinized blood of healthy donors by centrifugation over Ficoll/Hypaque (Pharmacia, Uppsala, Sweden) density gra~lients [3]. Human TIL cultures were derived from metastatic melanoma patients as previously described [31 ]. Growth medium for TIL cultures was either RPMI-1640 (Biofluids, Rockville, Mal.) containing 10 mM HEPES (Biofluids), 10% heat-inactivated human AB serum (PelFreeze Biologicals, Rogers, Ariz.), and 20% 4-day autologous/allogeneic lymphokine-activated killer (LAK) cell culture supematant, or AIM-V serum-free medium (Gibco, Grand Island, NY). Both media were supplemented with 2 mM glutamine, 50 1V/tal penicillin, 50 gg/tal streptomycin (NIH Media Unit, Bethesda, Md) and 1000 U/ml recombinant IL-2 (rlL-2; kindly supplied by Cetus Corporation, Emeryville, Calif.). Cultures were plated at a density of (2.5-5) x l0 s cells/ml and fed weekly by diluting the cell concentration with fresh medium. Standard tissue-culture chanabers, including 24-well plates, 6-well plates or T-75 flasks (Costar, Cambridge, Mass.) were incubated in a humidified 5% CO2 atmosphere at 37 ° C. Preparation o f L A K cell supernatants. PBMC derived from patients or normal donors were cultured at a concentration of 106 cells/ml in RPMI1640 medium supplemented with 2% human serum, antibiotics, glutamine and HEPES buffer and 1000 U/ml rIL-2. After incubation for 3 - 4 days in a humidified 5% CO2 atmosphere at 37 ° C, supernatants were collected, filtered through a 0.2 gm filter (Nalge, Rochester, N.Y.) and stored at 4° C until used as a supplement to the growth medium. Phenotype analysis. Cell-surface antigens were detected using flow cytometry on a fluorescence-activated cell sorter (FACS) 440 or FACScan (BectonDickinson). Fluorescein-isothiocyanate- (FITC)-conjugated antibodies (mAb) (Becton Dickinson) included: anti-Leu4 (CD3), antiLeu3a, (CD4), anti-Leu2a (CD8), and anti-Leul la (CD16). Phycoerythrin-conjugated antibodies included: anti-Leu3a (CD4), anti-Leul9 (CD56), and anti-Leu 15 (CD 1 lb). Washing and staining were performed in Hanks balanced salt solution (HBSS) without phenol red, containing 5% heat-inactivated fetal-calf serum (Biofluids, Rockville, Md.) and 0.03% sodium azide. Approximately 106 pelleted cells were stained with 15 gl diluted antibodies at 4 ° C for 30 min, washed twice and resuspended in 0.5 ml staining buffer for FACS analysis. Double-staining analysis was performed by simultaneous incubation of FITC- and phycoerythrinconjugated antibodies. Separation of PBMC and TIL cultures into CD4 +and CD8 +celI subsets. For positive panning, 6 × 107 fresh PBMC were incubated at room temperature with 3.5 ml 0.5% heat-inactivated human immune globulin (Armour Pharmaceutical Company, Ill.) and then introduced into T-25 polystyrene flasks (Corning, N.Y.) coated with covalently attached anti-CD4 or anti-CD8 mAb (kindly supplied by Applied Immune Sciences Inc., Menlo Park, Calif.). After 1 h incubation at room temperature, the suspension of unbound cells was removed and 5 ml "release medium" containing RPMI1640 supplemented with 10% heat-inactivated human AB serum, 10 mM HEPES, 50 IU/ml penicillin, 50 gg/tal streptomycin, 0.1 gg/ml phytohemagglutinin (PHA; purified HA 16/17 Wellcome, England) and 1000 U/ml rIL-2 was added. After 72 h of culture, cells were removed

from flasks by pipetting and replated at ( 0 . 5 - 1 ) x 106 cells/ml for 5 - 1 2 days in fresh medium containing 10% release medium without adding fresh PHA. Phenotypic analysis was carried out on days 8 - 1 2 post-separation. If the cultures contained more than 2% contamination of the irrelevant T cell subset population or double-positive CD4/CD8 cells, the cell population was further purified by a negative selection using the appropriate mAb [Anti-Leu3a (CD4), anti-Leul9 (CD56) Becton Dickinson, or anti-OKT8 (CDS) Ortho Diagnostic Systems Inc., N.J.] and sheep-anti-(mouse IgG)-coated magnefic beads (Dynabeads M-450, Dynal Inc., Fort Lee, N.J.). A sample of 10 7 w a s pelleted and then resuspended in 100 gl undiluted azide-free mAh at 4 ° C for 30 min. After washing twice at 4 ° C, cells were mixed with magnetic beads at a bead: target cell ratio of 40 : 1 in a total volume of 200 gl 2.5% human serum albumin (Armour Pharmaceutical Company, Ill.) in HBSS without Ca+/Mg2+. After centrifugation at 300 g and incubation at 4°C for 30 min, the mixture was diluted in 5 ml HBSS, and magnetic beads with or without coated cells were removed by a Dynal Magnetic Particle concentrator (Dynal MPC 1). The nonadherent cell population was retrieved, washed, and further tested by FACS analysis to confirm depletion of undesired cells. After purification, CD4 + and CD8 + cell subsets derived from PBMC were grown in the presence of 1000 U/tal IL-2 in either RPMI-1640 containing 10% human AB serum or AIM-V serumfree medium. Separation of TIL cultures into CD4 + and CD8 +cells was carried out as described above with the following modifications: (a) TIL cnltures were established in an exponential phase of growth before cells were subjected to separation; (b) release medium was supplemented with 20% LAK culture supernatant rather than PHA. The degree of purification of PBMC- and TIL-derived CD4 + cell subsets was generally >90% after the positive panning selection and >99% after the immunomagnetic bead negative selection. Purification of the CD8 + cell subset was >96% and >99% respectively. Purified CD4 + and CD8 + cell subsets derived from TIL were cultured in RPMI-1640 containing 10% human AB serum, 20% LAK cell supernatant, 1000 U/ml IL-2 and 1000 U/tal rhlL-4 (Immunex, Seattle, Washington). Transduction and G418 selection. During this study two retroviral vectors were used: N2 [7] and LNL6 [2], both carrying the bacterial gene for neomycin resistance (necY) and produced by the PA317 packaging cell line [20]. In the LNL6 vector, MoMuLV sequences containing gag and psi signals of the N2 vector were replaced by a region of Moloney murine sarcoma virus with several modifications that were designed to reduce reeombination events and prevent the production of a helper virus. Unless otherwise stated the LNL6 vector was used. In all the experiments presented, both vectors were free of helper virus. Cells in the exponential phase of growth were exposed to two sequential 2- to 6-h incubations with producer cell line supernatant containing the LNL6 or N2 vector (average vital titer: 106 G418-resistant colony-forming units/ml; multiplicity of infection: 4 - 8 ) in the presence of 5 gg/tal protamine sulfate (Eli Lilly and Co., Indianapolis, Ind.) at 37 ° C. An aliquot of cells was subjected to selection for 10 days 2 - 6 days after transduction by adding 0.3-0.4 mg/ml (active weight) G418 (Gibco) to the culture medium. Controls of nontransduced cells that were run in parallel all died at this concentration of ding. DNA analysis. DNA was isolated and digested with the SacI restriction enzyme, which cuts once in each of the long terminal repeat sequences of the LNL6 vector, releasing a 3.1-kb fragment containing the neo R gene. Samples of digested DNA (15 gg/lane) were electrophoresed in 0.8% agarose, and the separated fragments were transferred to nitrocellulose membranes and hybridized overnight at 42 ° C with a »2p-random-primerlabeled 0.9-kb Pst fragment of the neo ~ gene (specific activity, >108 dpm/gg) in 6 x standard saline citrate (SSC) 10% dextran sulfate/ 50 mM TRIS/HC1, pH 7.5/1 mM EDTA containing denatured salmon sperre DNA at >100 ~tg/ml. Blots were washed three times in 1 ×SSC (1 x is 0.15 M NaC1/0.015 M sodium citrate, pH 7) for 90 min, with a high-stringency wash in 0.1 x SSC at 55°C for 30 min. Autoradiography was carried out at -70 ° C using Kodak XAR-5 films with an intensifying screen. DNA digested with HindIII was used as size marker for the DNA fragments.

344 Table 1. Growth rate of nontransduced (NV), and transduced selected (LNL6-G) TIL TIL no.

Day of ~ansduction

Growth post-transduction Tirne Posttransduction (days)

740 659 830 660 816 844 811 822 884

24 30 13 33 27 20 14 21 15

29 28 35 35 35 37 35 32 24

Cumulative expansion indexa NV

LNL6-G

130 232 1Õ30 3 097 4549 14844 634 437 22

84 17.4 1809 680 1463 997 10.4 0.2 1.47

Cumulative expansion index was determined by calculating the final theorectical cell yield compared to the number of cells at the day of transduction

Detection ofneophosphotransferase activity. The assay was carried out as was previously described [24] with some modifications. Briefly, crude cell lysates of (2- 4) x 106 cells prepared by multiple freeze/thaw cycles, were electrophoresed on a non-denaturing sodium dodecyl sulfate/polyacrylamide gel. The washed gel was then overlayed with agarose containing kanamycin (25 ~tg/ml) and 1-2 nM [y-32P]ATP (6000 U/mmol) in buffer and incubated for 1 h at room temperature. Diffusable products were then transferred to Whatman P81 paper, which was washed twice with 50 mM sodium phosphate pH 7, at 80° C, and again with 80°C distilledwater. Autoradiographywas done as has already been described. Neophosphotransferase activity was considered positive if a band representing 32pqabeled kanamycin phosphate was seen at the same molecular mass position as the standard, a lysate of 105 cells of N7 (an N2-transfected G418-selected PA3 l 7 producer line).

Four-day proliferation assay in the presence of G418. Aliquots of 105 cells were plated in a flat-bottom 96-well plate (Costar, Cambridge, Mass.) and cultured for 4 days in the presence of 1000 U/tal IL-2 and various concentrations of G418 (0-1 mg/tal active weight) at a total volume of 200 ~tl. A 2-gl sample of [3H]thymidine (3.0 TBq/mmol, Du Pont, New Products, Boston, Mass.) were added for the last 18 h of incubation. Harvesting and counting were carried out by using an LKB Wallac Betaplate (Pharmacia/LKB Sweden).

Limiting-dilution cultures. Varying numbers (10 2 to 3 x 104) of cells were plated in 96 round-bottom microtiter wells (Costar, Cambridge, Mass.) at a total volume of 200 gl AIM-V containing 1000 U/ml rIL-2. Each well contained 105 irradiated (10 000 rad) Epstein-B arr-virus-transformed B cells as feeders. For each cell dilution 24 wells were plated with or without 0.3 mg/tal G418. Cell growth was evaluated on day 14 after plating. Controls of irradiated reeder cells with and without G418 were run in parallel and were negative on day 14. As described elsewhere [19] the frequency of cell growth and the correlation coefficient of the regression analysis for the Poisson distribution were calculated by plotting the percentage of nongrowing cultures at each cell dilution against the number of cells originally plated in the wells.

Results Effect of LNL6 transduction and G418 selection on TIL growth H u m a n m e l a n o m a T I L cultures in their e x p o n e n t i a l phase o f g r o w t h were transduced with the retroviral vector L N L 6 carrying the bacterial neo R gene. Aliquots o f nontransd u c e d and t r a n s d u c e d cells were selected by e x p o s u r e to G418 in culture.

G r o w t h o f nontransduced, and t r a n s d u c e d G418-selected ( L N L 6 - G ) cells f r o m nine T I L cultures, expressed as c u m u l a t i v e e x p a n s i o n indices, are shown in Table 1. Representative g r o w t h curves o f two T I L cultures are shown in Fig. 1. The rate o f proliferation of nontransduced T I L derived f r o m different individuals v a r i e d significantly (range o f 2 2 - 1 4 844 c u m u l a t i v e e x p a n s i o n index; m e d i a n 634) over 2 4 - 3 8 days after transduction. The relatively low e x p a n s i o n indices o f t r a n s d u c e d - s e l e c t e d ( L N L 6 - G ) cultures (range: 0 . 2 - 1 8 0 9 , m e d i a n 84) during this same time p e r i o d reflected the decrease in cell n u m b e r occurring during G418 selection. M o s t o f the L N L 6 - G cultures res u m e d exponential g r o w t h as soon as the cells expressing the neoR gene b e c a m e d o m i n a n t (days 8 - 1 0 in G418). A l l T I L d e r i v e d f r o m n o n t r a n s d u c e d cultures died after 10 days in 0.3 m g / t a l G418 (data not shown). In three L N L 6 - G cultures (811, 822 and 884), an exponential growth was not o b s e r v e d even after G418 removal. In one out o f these three cultures the low e x p a n s i o n index correlated with a l o w rate of g r o w t h o b s e r v e d with the appropriate n o n t r a n s d u c e d cells (TIL 884). In all three cultures there was e v i d e n c e for the p r e s e n c e o f the neoR gene as j u d g e d b y detection o f n e o p h o s p h o t r a n s f e r a s e activity (see Fig. 2 B for TIL 884, data not shown for T I L 811 and 822). In order to ensure that gene insertion had not caused an a u t o n o m o u s growth o f the t r a n s d u c e d TIL, cultures were m a i n t a i n e d without I L - 2 and were f o l l o w e d for cell counts and D N A synthesis. The results s h o w e d that l o n g - t e r m proliferation o f t r a n s d u c e d selected ceUs was I L - 2 - d e p e n dent. A representative e x p e r i m e n t o f T I L t r a n s d u c e d with the N 2 vector (a similar construct to L N L 6 ) is shown in Table 2. There was no e v i d e n c e for D N A synthesis in N 2 - t r a n s d u c e d selected cell samples taken at various time intervals ( 7 - 4 5 days) after 1L-2 withdrawal. T w o TIL cultures were f o l l o w e d for a p e r i o d o f m o r e than 180 days and there was no e v i d e n c e for proliferating cells in cultures without IL-2 as m e a s u r e d b y trypan blue exclusion (data not shown). In s u m m a r y , t r a n s d u c e d T I L in m o s t cases could be selected in G 4 1 8 - c o n t a i n i n g m e d i u m under conditions in w h i c h only t r a n s d u c e d cells survived. T r a n s d u c e d selected cells did not change their g r o w t h rate and m a i n t a i n e d their IL-2 d e p e n d e n c e in l o n g - t e r m culture.

345 CD4 + TIL

CD8 + TIL

10 m

09

O NV Z& LNL6 [ ] LNL6-G

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Days Post Transduction Fig. 1. Growth curves of nontransduced (NV), transduced nonselected (LNL6) and transduced G418-selected (LNL6-G) cells derived from TIL culture 816 (A) and TIL 844 (B). G418 selection for TIL 816 and TIL 844 was on days 5 - 16 and 6 - 17 post-transduction, respectively

1

2

3

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1

2

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3.1 k b - II

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TIL #660

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Fig. 2 A - C. Evidence for the presence of the neo e gene in representafive

samples of nontransduced (NV) transduced nonselected (LNL6) and transduced G418-selected (LNL6-G) TIL. A Southem blot analysis in nontransduced [10, 31] and LNL6-G [1, 21] of TIL740 [10, 21] and TIL 844 [1, 31] was performed on days 22 and 37 post-transduction, respectively. SacI-digested DNA, probed with the neoR gene released a single band of the expected 3.1 kb size. B Detection of neophosphotransferase

(NPT) activity by measuring the phosphorylation of kanamycin with [32p]ATR Samples of NV [1, 10], LNL6 [21, 25] and LNL6-G [28, 31] TIL 659 [10, 21, 31] and TIL 884 [1, 25, 28] were taken on days 28 and 24 post-transduction, respecdvely. N7 is the positive control. C Four-day proliferation assay in the presence of G418. Samples of TIL 660 and TIL 816 were taken on days 17 and 15 post-transduction, respectively

346 Table 2. Effect of interleukin-2 (IL-2) withdrawal on nontransduced and transduced selected TIL growtha Day ofassay

[3H]Thymidine uptake (cpm _+SE)

Culture

wi~outIL-2 7

16

24

45

wi~IL-2

Medium NV N2G

393 + 63 439+_ 27 720 4-_ 4

35396+- 813 26 395 + 2216

Medium NV N2G

135+- 27 393+- 63 210+- 9

74576+-6293 63 143___9814

Medium NV N2G

ND 166+- 28 78+- 8

43 954+-6408 54972+3525

Medium NV N2G

298 +- 56 263 + 107 172+- 59

101 156+-3773 52411 +2701

of G418. On average 10%-18% of the LNL6 transduced nonselected TIL could proliferate in 0.8-1 mg/ml G418 while >98% of the nontransduced TIL died in these concentrations. Figure 2 C shows two representative samples of 4-day proliferation assays of transduced TIL. In summary, LNL6-transduced TIL contained the vector DNA, expressed the enzymatic activity of the gene product and could proliferate in high concentrations of G418 that were highly toxic to nontransduced controls.

Effect of transduction and selection on ceIl-surface phenotypes

a Nontransduced (NV) and N2-transduced G418-selected (N2G) TIL were washed three times on day 14 post-lransduction and cultured with or without IL-2 at 1 x 106 cells/ml in bulk cultures. Samples containing 1 × 105 cells were taken at various intervals and pulsed with [3H]thymidine for 18- 24 h before harvesting

Evidence for the presence of the neoR gene in LNL6-transduced TIL Southem blot analysis confirmed the presence of a grossly intact neoR gene in LNL6-transduced TIL. Two representative samples (740 an 844) are shown in Fig. 2A. A single band of the expected 3.1 kb size that hybridized with the neoR probe was present in the transduced G418selected TIL but was not seen in nontransduced cells. The enzymatic activity of the neophosphotransferase gene product was demonstrated in all samples of transduced unselected LNL6 TIL. The specific activity was higher in the G418-selected cultures (LNL6-G), whereas no activity was seen in nontransduced TIL. Figure 2 B shows neophosphotransferase activity in two representative population samples of transduced TIL. In a 4-day proliferation assay, cells were cultured in the presence of 0 - 1 mg/ml G418 and transduced selected TIL expressed sufficient neophosphotransferase to become resistant to high concentrations

FACS analyses were performed on five populations of transduced selected (LNL6-G) TIL and their non-transduced controls (Table 3). TIL isolated from different donors have individual phenotypic profiles as defined by various cell-surface determinants. TIL from two donors, with a predominantly CD8 ÷ phenotype in the original culture (>95% CD8 ÷ cells), maintained their phenotypic profiles after the transduction/selection procedures. In contrast, two separate TIL cultures, transduced and selected independently but derived from one tumor specimen (798), started with a predominance of CD8 + cells but then showed an increased percentage of CD4+ cells in the selected cultures. Of two cultures beginning with a mixed population of CD4+ and CD8 ÷ cells, one culture (816) showed a slight shift towards more CD4 ÷ cells (76% to 89%) while the other TIL culture (830) showed a marked shift towards more CD4 ÷ cells (56% to 90%) in the transduced G418-selected population. No major changes in expression of CD3, CD16, CD56 and CD1 lb cell-surface markers were observed.

Transduction of TIL cultures with a mixed population of CD4 + and CD8 + cells To study further the transduction of individual T cell subsets, three melanoma TIL cultures with mixed populations of CD4 ÷ and CD8 + cells (CD4/CD8 cell ratios of 2-4) were transduced with the LNL6 vector. After 6-13 days (without selection), the cells were separated into purified CD4+ and CD8 + cell subsets for individual analysis. Phenotypic profiles of a representative TIL culture are

Table 3. Phenotypic analysis of transduced selected (LNL6-G) vs nontransduced (NV) TIL cultures TIL no.

740 660 798 a 798 b 816 830

Time posttransduction (days) 19 21 36 28 27 27

Positive cells (%) CD3

CD4

CD8

CD16

CD56

CD1 lb

NV

LNL6-G

NV

LNL6-G

NV

LNL6-G

NV

LNL6-G

NV

LNL6-G

NV

LNL6-G

99 94 93 88 92 87

99 92 95 87 98 84

0.3 0 2 6 76 56

0.3 0.5 40 43 89 90

99 95 92 82 21 22

98 92 57 35 12 0.8

2 0 0 0 0 0.6

4 0.8 1 0.5 2 0

0 7 0 38 2 0

0 6 0.5 7 0 0

14 0 0 0.7 0.4 0

7 0.2 0.6 0 0 0

347 Table 4. Phenotypicprofiles of transduced unseparated and transduced purified CD4+ and CD8+cell subsets Phenotypic marker

Positive cells (%) Pre-transductiorê unseparated

26 days post-transductionb Purified

Unseparated

CD4+/CD8CD4-/CD8+ CD4+/CD8+ CD56+/CD3CD56-]CD3+ CD56+/CD3+

59 20 17 1 87 11

NV

LNL6

LNL6-CD4+

LNL6-CD8+

26 66 3 0 85 12

35 56 3 0 88 11

97 0 0 0 98 0

0 99 0 0 98 0

a,b A representative double-staining FACSanalysis of TIL 913 was done on the day of transduction a and 26 days after transduction b. TIL 913 was transduced on day 27 of culture and separated into CD4+and CD8+cells subsets (LNL6-CD4+, LNL6-CD8+) 12 days after transduction. Double-

positive CD4+/CD8+ cells were removed from both T cell subsets by immunomagnetic beads (see Materials and methods). Controls of nontransduced (NV) and transduced unselected (LNL6) cells were run in parallel

shown in Table 4. This TIL culture, consisting of 59% CD4+/CD8 -, 20% CD4-/CD8 + and 17% CD4+/CD8 + cells was transduced 12 days before sepm'ation into purified CD4+/CD4- and CD8+/CD4- cell subsets. FACS analysis done 26 days after transduction (14 days after separation) revealed that, with time, these cultures increased the percentage of CD8 + cells and decreased CD4 + cells regardless of whether or not they were subjected to transduction. The isolated purified CD4+ and CD8 + cell subsets, however, maintained their phenotypic profiles. Similarly, in two other nontransduced and transduced unseparated TIL cultures (data not shown) some fluctuations in the CD4/CD8 cell ratio have occurred with time, while the separated transduced CD4+ and CD8 + subsets have maintained their purified phenotypic profiles. Evidence for transduction of the unseparated and the isolated purified CD4 + and CD8 + cells was shown by detection of neophosphotransferase activity. In each of the three TIL culture specimens there was evidence for the gene product in the transduced unseparated as well as in the transduced CD4 + and CD8 + cell subsets but no such activity was evident in the nontransduced cells. Thus both CD4 ÷ and CD8 + cells were transduced by this retroviral vector. In order to compare the frequency of transduced cells in the purified CD4 + and CD8 ÷ cell subsets, limiting-dilution cultures were carried out in the presence and absence of G418. Results of a representative analysis are shown in Table 5. In the absence of G418, the frequencies of cell growth of nontransduced and transduced unseparated cells were 1/1047 and 1/1239 respectively. Under the selective pressure of G418, no growth was observed in the nontransduced culmres, while a frequency of 1/20076 cell growth was demonstrated in the transduced unseparated culmres. The cell growth frequencies of CD4 ÷ and CD8 ÷ cell subsets were almost the same in the absence (1/1564 and 1/1456, respectively) or presence (1/18 039 and 1/15584, respectively) of G418. Thus, only about 6 % - 9 % of the cells in cultures exposed to the retroviral vector could grow in G418 after transduction. Of these, both T cell subsets could be transduced within a heterogeneous T cell population. Preferen-

tial transduction of either CD4+ or CD8 +cells could not be demonstrated under these experimental conditions.

»ansduction and selection of purified CD4 + and CD8 + TIL subsets Purified populations of CD4+and CD8 + cells (>96%) were isolated from three different TIL cultures and were then transduced with the LNL6 vector. Two of the TIL cultures were derived from metastatic melanoma (TIL 740, 900) and orte TIL culture was derived from a primary lung cancer (TIL 761). Nontransduced, transduced (LNL6) and transduced G418-selected (LNL6-G) cells from each of the purified subsets were followed for growth and tested for evidence for the presence of the neoR gene. Figure 3 shows the growth curves of CD4+ and CD8 + cells derived flora the two melanoma TIL. Purified CD4+ cell subsets derived from TIL 740 proliferated rapidly while the isolated CD8 + cell subsets grew at a slower rate. All groups of cells derived from TIL 900 proliferated rapidly while a slower rate of proliferation was observed in the CD4+ transduced

Table 5. Cell growth frequencies of transduced TIL-derivedCD4+ and CD8+ cellsa Cells

NV LNL6 LNL6-CD4+ LNL6-CD8÷

Growth frequency Without G4 l 8

With G418

1/1047 111239 1/1564 1/1456

0.96

348

A

B

1013 1012 0 Õ

10TM 1013

O NV A LNL6 [] LNL6-G

lO 1°

~

~~

1012

0-..,....43

1011

.&...~~"

o.. ~" 10~°

.13 E

109

:3

108!

109

.~'7

lO~ 1060

lo] I

I

I

I

1

I

I

I

I

7

14

21

28

35

42

49

56

63

6[

70

10 I

_/"J~

"~'I° I

I

I

L,

6

12

18

24

3

0

I

I

L

I

36

42

48

54

60

Days Post Transduction Fig. 3. Growth curves of nontransduced (NI0, transduced nonselected (LNL6) and transduced G418-selected (LNLó-G) cells derived from CD4 + and CD8 + cell subsets of TIL 740 (A) and TIL 900 (B). G418 selection for TIL 740 subsets and TIL 900 subsets was on days 5 - 1 5 and 5 - 1 4 post-transduction, respectively

1

2

3

4

N?

1

2

3

/-,

5

6

NPT ii'~~Ji~! 3.1kb--

A

B 120 110

;D4+ TIL

1001 B 90 ~" ~ "~"~" 1~ 80 "O"~ t-

70

"~ ep-

50 40

13

-b co

CD8 + TIL

O NV ZXLNL6 ~ -O-O E]LNL6-G ~

""O"~

,~.~ . . . . O . _

~

__O __ __

" , . , . , .... ~..>«..~._

_.. O

6O

30 20

04 lo! - ~ ~ , - ~ " ,~ ~ - ~, - , 4 0.0

C

0.2

0.4

0.6

0.8

1.0

1.2 0.0

i

I

0,2

0.4

~

0.6

o

0.8

1.0

1.2

G418 Concentration mg/ml

Fig. 4 A - C. Evidence for the presence of the neo« gene in TIL-derived CD4 + and CD8 + representative samples of nontransduced (NV), transduced nonselected (LNLó) and transduced G418-selected (LNL6-G) cells. A Southem Not analysis of NV [10, 31] and LNL6 [1, 21] of CD4 + [ 10, 21] and CD8 + [ 1, 31 ] cells derived from TIL 740 was done on day 29 post-transduction. SacI-digested DNA probed with the neoR gene released a single band of th expected 3.1 kb size. B Detection of neophos-

photransferase (NPT) activity by measuring the phosphorylation of kanamycin with [3ap]ATR Samples of NV [1, 10], LNL6 [21, 25] and LNL6-G [28, 31] of CD4 + [10, 21, 31] and CD8 + [1, 25, 28] cells derived from TIL 900 were taken on day 24 post-transduction. N7 is a positive control. C Four-day proliferation assay in the presence of G418. Samples were taken from TIL 900 cell subsets on day 35 post-transduction

G418-selected cultures. The growth rate of the CD4 + and CD8 ÷ cell subsets derived from TIL 761 (lung cancer) followed the pattern of melanoma TIL 740 (data not shown). The presence of the n e o R gene was demonstrated by Southem blot analysis, detection of neophosphotransferase activity and by the ability of the cells to proliferate in the presence of various concentrations of G418. A 3.1-kb

neoR-hybridizing fragment was present in the transduced nonselected CD4 ÷ and CD8 ÷ cells but not in the nontransduced control cells (Fig. 4A). The enzymatic activity of the gene product was evident in CD4+ transduced nonselected cells and was stronger in the CD8 + transduced nonselected cells. Neophosphotransferase specific activity was further enhanced in the transduced G418-selected cells from both CD4 + and CD8 + cells (Fig. 4B). In 4-day pro-

349 Table 6. Phenotype profiles of melanoma TIL-derived CD4+ and CD8+ cell subsets before and after transduction Phenotypic marker

Positive cells (%) TIL cultured pm-separationa

CD4+ cell subset

CD8+ cell subset

Pre-trans- Post-transductioncductionb NV LNLó

LNL6-G

Pre-trans- Post-transduction° ductionb NV LNL6

LNL6

CD4+/CD8 CD4/CD8 + CD4+/CD8+ CD56+/CD3CD56/CD3 ÷

27 58 4 12 84

96 0 2 0 92

99 0 0 0 99

99 0 0 0 99

98 0 0 0 99

0 99 0 0 97

0 99 0 0 62

0 99 0 0 71

0 99 0 0 76

CD56+/CD3+ CD16+

3 4

4 0

0 4

0 3

0 2

2 0

37 3

28 2

23 5

a c A representative double-staining FACS analysis of TIL 740: a before separation into CD4+ and CD8+ cell subsets, b CD4+ and CD8+ cell subsets at day of transduction, ° CD4+ and CD8+ cell subsets on day 20 post-transduction. Controls of nontransduced cells (NV) were run in parallel with transduced nonselected (LNLó) and transduced G418-selected (LNL6-G) cells

Table 7. Phenotype profiles of PBMC-derived CD4+ and CD8+ cell subsets before and after transduction Phenotypic marker

Positive cells (%) CD4÷ cell subset Pretransductiona

CD4+/CD8CD4/CD8 + CD4+/CD8+ CD56+/CD3CD56-/CD3+ CD56+/CD3+ CD16

100 0 0 0 99 0 0

CD8+ cell subset Post-transductionb

Pretransductiona

NV

LNLó

LNL6-G

99 0 l 0 97 2 5

99 0 1 0 97 1 0

96 0 2 0 96 2 3

0 99 0 0 93 7 0

Post-transductionb NV

LNL6

LNLó-G

0 95 2 0 52 47 3

0 94 2 0 48 51 3

0 96 1 0 73 26 1

a. b A representative double-staining FACS analysis of PBMC 4 done on day of transductiona and on day 16 post-transductionb. Controls of nontransduced cells (NV) were run in parallel with transduced non-selected (LNL6) and transduced G418-selected (LNL6-G) cells

liferation assays CD8÷-transduced nonselected cells were more resistant to G418 than were CD4+-transduced nonselected cells (Fig. 4C). Although only a small proportion of CD4 + transduced nonselected cells could proliferate in high concentrations of G418 in the 4-day proliferation assay, we could have selected CD4 ÷ transduced cells in long-term growth in the presence of 0.3 mg/1 G418. Phenotypic analysis of culture g r o w n from the purified CD4 + and CD8 + cell subsets showed that each subset m a i n t a i n e d its phenotypic profile after b e i n g transduced and selected in m e d i u m c o n t a i n i n g G418. The CD8 + cell subsets assayed 20 days after transduction contained a high proportion of double-positive CD3+CD56 + cells but 99% of them were CD8 + cells (Table 6). Thus, both TIL-derived purified C D 4 + and CD8 + cell subsets were able to be successfully transduced with the neo R gene. Furthermore, all cell subsets m a i n t a i n e d their purified p h e n o t y p i c profiles in long-term culture after transduction.

Transduction and selection o f P B M C - d e r i v e d CD4 + and CD8 + cells The previous studies demonstrated transduction of CD4 + and CD8 ÷ TIL. To study further the transducibility of T cell subsets in general, purified populations of CD4+and CD8 ÷ cells derived from the P B M C of five n o r m a l blood donors were subjected to transduction by the L N L 6 vector. In most cases, the purified CD4 + cell subsets proliferated rapidly while m u c h slower growth was observed in the purified CD8 + cell subsets. N o n t r a n s d u c e d control cells, transduced (LNL6) and transduced G418-selected (LNL6-G) cells from each of the purified subsets were tested for evidence for the presence of the neo R gene. Representative data (PBMC 4) are shown in Fig. 5. The expected 3.1-kb neoR-hybridizing fragment was present in the transduced nonselected CD4 ÷ and CD8 + cells but was not in n o n t r a n s d u c e d cells (Fig. 5 A). The enzymatic activity of the gene product (neophosphotransferase) was evi-

350 1

2

3

N7

L

3.1kb--

NPT--

A

B CD4 + Cells

1

2

3

4

0 0 8 + Cells

120 110 1001 - ~ ~ Ek. 80 ~ , ,

E

E c-

F-

#

40~

~-~3 ~

70 ~ 60 \ ' ~ . 50 \~,

O NV ALNL6 n LNL6-G

30

20

o~

C

10 0 0.0

-0.2

0.4

0.6

- ....

,,~. . . . . o 0.8

--& o 1.0

.2 0.0

0.2

0.4

0.6

0.8

10

1.2

G418 Concentration mg/ml

Fig. 5A-C. Evidence for the presence of the neoR gene in CD4+ and CD8+ representative cell samples of nontransduced (NV), transduced nonselected (LNL6) and transduced G418-selected (LNL6-G) cells derived from PBMC 4. A Southern Not analysis of NV [10, 31] and LNL6 [1, 21] of CD4+ [10, 21] and CD8+ [1, 31] cells was done on day 16 post-transduction. SacI-digested DNA probed with the neo R gene

released a single band of the expected 3.1 kb size. B Detection of neophosphotransferase (NPT) activity by measuring the phosphorylationof kanamycin with [32P]ATRSamples of NV [10, 31] and LNL6 [21, 1] of CD4+ [10, 21] and CD8+ [1, 31] cells were taken 22 days post-transduction. N7 is a positive control. C Four-day proliferation assay in the presence of G418. Samples were taken on day 16 post-transduction

dent in purified CD4 + and CD8 ÷ transduced nonselected cells while no such activity could be detected in nontransduced cells (Fig. 5 B). CD8 + cells subsets were more resistant than CD4 +cells to high concentrations of G418 (Fig. 5 C). In spite of the low percentage of [3H]thymidine uptake of CD4+ transduced nonselected cells, there was evidence of the presence of the gene-product enzymatic activity and CD4 ÷ transduced cells could be selected in long-term growth. In high concentrations of G418 ( 0 . 8 1 mg/ml) CD4 ÷ and CD8 ÷ transduced nonselected cells displayed 10% and 70% [3H]thymidine uptake, respectively. Transduced selected cells of both CD4 + and CD8 + subsets displayed high thymidine uptake in all G418 concentrations tested while no thymidine uptake was observed in the nontransduced cultures when exposed to high concentrations of G418. Phenotypic analysis performed more than 1O days after transduction showed that the purified CD4 + and CD8 + cell subsets maintained their phenotypic profiles in the nontransduced, transduced nonselected as well as in the transduced G418-selected cultures. A representative analysis is shown in Table 7. In summary, in both PBMC-derived CD4+ and CD8 ÷ cells there was evidence for the presence of the n e o R gene and cell subsets maintained their phenotypic profiles after transduction.

Discussion Genetic modification of human TIL was performed using an amphotropic murine retroviral vector (LNL6 or N2) containing the n e o R gene. The presence of the n e o R gene in the D N A of TIL populations was shown by Southern blots, detection of the gene product activity (neophosphotransferase) and by the ability of TIL to proliferate in high concentrations of G418. Since the TIL culmres used for the adoptive immunotherapy of cancer in humans offen contained a mixed population of CD4 ÷ and CD8 ÷ cells [1, 10, 21, 31] prior to gene transduction, it was important to test whether these cell subsets showed differential susceptibility to the transduction procedure. In this study we have shown that CD4 + and CD8 + T cells containing the n e o R gene could be recovered after exposing either heterogeneous TIL populations or purified TIL- and PBMCderived CD4 ÷ and CD8 ÷ cell subsets to the retroviral vector. Murine retroviruses integrate randomly into the host genome [30] and inactivation of genes with essential functions or activation of inappropriate genes in at least some cells is a possibility [33]. Therefore in our study, the growth rate and IL-2 dependence as well as phenotypic profiles were carefully followed in long-term cultures of

351

transduced TIL to confirm whether normal gene functions were maintained after transduction. The presence of the neo R gene in transduced G418-selected TIL populations did not alter their proliferation rate and their IL-2 dependence when compared to control cultures of nontransduced TIL. The variable growth rates displayed by various nontransduced and transduced cultures are consistent with previous reports that showed a variety of patterns of TIL proliferation in culture [31]. Fluctuations in the CD4/CD8 cell ratio seen in some transduced selected bulk TIL cultures in this report as well as in the previous report [14], were also seen in other samples of untreated TIL, and probably reflect clonal overgrowth in long-term bulk cultures. In order to verify that the phenotypic profiles of cells following transduction and during subsequent expansion in culture did not differ significantly from those undergoing expansion only, it was important to test highly purified cell populations in which a shift in cell-surface marker expression could not be a result of a selective clonal growth pressure. Thus, highly purified CD4 ÷ and CD8 + cell subsets (>98% purity) derived from either the same specimen of TIL or PBMC were transduced with the LNL6 vector and were subsequently followed after long-term growth. The presence of the neoÆ gene was evident in transduced nonselected cultures of both CD4 + and CD8 + cells. In each of the non-transduced, transduced nonselected and transduced selected purified cell subsets, the distinctive T cell surface markers were fully sustained after transduction and during long-term growth, suggesting that the insertion of the neo ~ gene into the host genome did not induce any change leading to alteration in the expression of T cell markers. These data also support the interpretation that phenotypic shifts observed in transduced selected TIL reflect a clonal overgrowth rather than induced genomic changes. Nishihara et al. [22] showed that the original phenotypic profile of a Thyl÷, Lytl-2+3 + murine cytotoxic T lymphocyte clone was also retained after transduction with a retroviral vector carrying the murine c~-interferon gene. Retroviral-mediated gene transfer is a highly efficient method for transferring genes into a variety of mammalian cell types [4, 5, 6, 9, 11, 12, 16, 18, 27, 29, 34]. Since TIL cultures are rapidly proliferating they may serve as good target cells for retroviral-mediated gene transfer. The integration of the LNL6 vector into the TIL genome could be detected in unselected and G418-selected cultures and was stable after long-term growth in culture. The frequency of transduction, assessed by linear regression analysis of limiting-dilution cultures 3 - 4 weeks after transduction, was 6%-9% in unselected cultures. This rate is comparable to the transduction frequency demonstrated immediately after transduction in precursors of murine cytotoxic lymphocytes [23]. In other studies, the efficiency of transduction, determined by the ability to grow in a selective medium or estimated from DNA analysis, varied from 4% to 30% [9, 13, 15]. In this study, we have shown that both CD4 ÷ and CD8 + cell subsets derived from PBMC or from bulk TIL cultures could be transduced by the LNL6 vector. Transduction of heterogeneous TIL populations containing both CD4+ and

CD8 + TIL resulted in neoR-gene-containing cells of both cell subsets, and the frequency of transduction, calculated by linear regression analysis of limiting-dilution cultures plated with or without G418, was almost identical in the isolated CD4+ and CD8 + cell subsets. These T cell subsets, however, were isolated 12 days after transduction and, during this time period, the differential growth rate of these subpopulations in the heterogeneous transduced culture might have led to a change in the proportion of transduced cells in these T cell subsets. In summary, we have shown that the retroviral LNL6 vector can reliably transduce cultures of heterogeneous TIL cells as well as isolated purified TIL-derived and PBMC-derived CD4+ and CD8 + cells. Clinical trials using neoÆ-marked TIL, as a part of adoptive immunotherapy protocols for the treatment of cancer, have already begun in an attempt to follow the distribution and survival of TIL in treated patients [26 a]. Because of the ability of TIL to home selectively to tumor foci [8], the easily transduced CD4+ and/or CD8 + cells might be further used as a delivery system for other genes (e. g. selected cytokine production), which would locally augment the anti-cancer activity of the effector TIL subset(s). The ability to transduce PBMCderived CD4 + and CD8 + cells efficiently may be of value in the study of the role of these T cell subpopulations in immunological reactions. Acknowledgements. We wish to thank Ann Stephen for her technical assistance, and Liliana Guedez and Ellen Bolton for performing all FACS analyses.

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