Patient eligibility

Patients had biopsy-proven recurrent, resected recurrent, or residual epithelial FR⁺ ovarian cancer that failed standard effective therapy, including cisplatin/carboplatin – or paclitaxel-containing regimens. Patients ranged in age from 33 to 60 years and had clinical Eastern Cooperative Oncology Group performance status of 0 or 1. Eligibility criteria required serum creatinine levels ≤1.6 mg/dL and bilirubin <2.0 mg/dL. Blood eligibility criteria included hemoglobin >9.0 g/dL, WBC >3,000/mm³, and platelets >100,000/mm³ and an intact immune system as evidenced by a positive reaction to Candida albicans skin test, mumps skin test, or tetanus toxoid skin test on a standard anergy panel.

Response assessment

Patients received radiologic evaluation by magnetic resonance imaging, computed tomography, or sonography immediately before treatment and at completion of therapy. Disease response was determined by comparison of pretreatment and post-treatment images. In addition, serum CA-125 levels were determined following treatment and compared with pretreatment CA-125 levels.

T cell generation

A detailed description of the generation and characterization of T cells used in cohort 1 of the study has been published previously (22). Briefly, patient PBMCs derived from leukapheresis were stimulated with anti-CD3 (OKT3, Ortho Biotech, Raritan, NJ) and human recombinant IL-2 (600 IU/mL; Chiron, Emeryville, CA). After 3 days of culture, ~5 × 10⁷ to 1 × 10⁸ lymphocytes were taken and transduced with retroviral vector supernatant (Cell Genesys, San Francisco, CA) encoding the chimeric MOv-γ gene and subsequently selected for gene integration by culture in G418.

For the generation of dual-specific T cells used in cohort 2, stimulation of T cells was achieved by coculture of patient PBMCs with irradiated (5,000 cGy) allogeneic donor PBMCs from cryopre-served apheresis product (mixed lymphocyte reaction). The MHC haplotype of allogeneic donors was determined before use, and donors that differed in at least four MHC class I alleles from the patient were used. Culture medium consisted of AimV medium (Invitrogen, Carlsbad, CA) supplemented with 5% human AB⁻ serum (Valley Biomedical, Winchester, VA), penicillin (50 units/mL), streptomycin (50 mg/mL; Bio Whittaker, Walkersville, MD), amphotericin B (Fungizone, 1.25 mg/mL; Biofluids, Rockville, MD), L-glutamine (2 mmol/L; Mediatech, Herndon, VA), and human recombinant IL-2 (Proleukin, 300 IU/mL; Chiron). Mixed lymphocyte reaction consisted of 2 × 10⁶ patient PBMCs and 1 × 10⁷ allogeneic stimulator PBMCs in 2 mL AimV per well in 24-well plates. Between 24 and 48 wells were cultured per patient for 3 days, at which time transduction was done by aspirating 1.5 mL of medium and replacing with 2.0 mL retroviral supernatant containing 300 IU/mL IL-2, 10 mmol/L HEPES, and 8 μg/mL polybrene (Sigma, St. Louis, MO) followed by covering with plastic wrap and centrifugation at 1,000 × g for 1 hour at room temperature. After overnight culture at 37°C/5% CO₂, transduction was repeated on the following day, and then medium was replaced after another 24 hours. Cells were then resuspended at 1 × 10⁶/mL in fresh medium containing 0.5 mg/mL G418 (Invitrogen) in 175-cm² flasks for 5 days before resuspension in media lacking G418.

Cells were expanded to 2 × 10⁹ and then restimulated with allogeneic PBMCs from the same donor to enrich for T cells specific for the donor allogeneic haplotype. Restimulation was done by incubating patient T cells (1 × 10⁶/mL) and stimulator PBMCs (2 × 10⁶/mL) in 3-liter Fenwall culture bags in AimV + additives and IL-2 (no G418). Cell numbers were adjusted to 1 × 10⁶/mL, and IL-2 was added every 2 days, until sufficient numbers for treatment were achieved.

Cell lines, flow cytometry, and IFN-γ secretion assay

Tumor cell lines used in assays of T cell function were the FR⁺ human ovarian cancer cell line IGROV-1 (23) and FR⁻ melanoma cell lines Mel 526, Mel 624, Mel 888, and Mel 1866 (Surgery Branch, National Cancer Institute, Bethesda, MD). Tumor cells were maintained in RPMI supplemented with 10% FCS (Invitrogen), penicillin (50 units/mL), streptomycin (50 mg/mL), amphotericin B (1.25 mg/mL), and L-glutamine (2 mmol/L). A melanoma-specific T cell line used in some experiments was derived from tumor-infiltrating lymphocytes of a patient at National Cancer Institute and maintained in T cell culture medium described above.

Expression of chimeric MOv-γ receptor by transduced T cells was determined using flow cytometry following staining with phycoerythrin-conjugated Id18.1, a monoclonal antibody specific for the MOv-18 idiotype (24). T cells were stained with phycoerythrin-conjugated mouse IgG1 as a control for nonspecific binding.

Transduced T cells were assessed for their ability to respond against the FR antigen by coculture with IGROV-1 ovarian cancer cells. T cells (1 × 10⁵) were incubated with IGROV-1 cells (1 × 10⁵; or FR-negative control tumor cells) in triplicate wells of 96-well plates. After overnight culture, supernatant was taken and assayed for IFN-γ using ELISA kits according to manufacturer’s instructions (Endogen, Woburn, MA). T cells in cohort 2 of the study were also assessed for their ability to secrete IFN-γ in response to allogeneic stimulator PBMCs freshly thawed from cryopreserved stocks. Anti-human CD3 (OKT3) was also used to stimulate T cells to gauge their maximal capacity to respond to TCR-CD3 engagement. OKT3 was immobilized on 96-well plastic plates at 0.5 μg/well in 100 μL PBS overnight at 4°C.

In some experiments, a 25% proportion of patient serum was included in T cell cocultures to determine possible effects of patient serum on T cell function. In some assays, protein G (Amersham Biosciences, Piscataway, NJ; 20 μL/mL) was added to serum before coculture and incubated for 1 hour at 4°C with gentle rocking to deplete patient serum of immunoglobulin.

Serum FR titer assay

A double-determinant assay was done essentially as described (25) using MOv19, a non–cross-reacting antibody directed against FR (18), as catcher. Briefly, 96-well flat-bottomed maxisorp plates (Nunc, Roskilde, Denmark) were coated with 200 μL of MOv19, at 1 μg/mL in PBS, and incubated overnight at 4°C. Plates were washed and blocked for 1 hour with 200 μL/well of 0.5% bovine serum albumin in PBS; 100 μL of sample was added to wells. A positive control consisted of tissue culture supernatant from IGROV-1 cells. Plates were incubated 2 hours and washed, and 100 μL of biotinylated MOv18 (0.25 μg/mL) were added followed by incubation at room temperature for 2 hours. Plates were washed, and streptavidin-horseradish peroxidase in PBS/0.5% bovine serum albumin was added 100 μL/well and incubated for 0.5 hour. Plates were washed, and 100 μL/well trimethylbenzidine was added and incubated for 5 to 10 minutes, and reaction was stopped with 1 mol/L H₂SO₄. Plates were read on spectrophotometer 450 nm within 0.5 hour of stopping reaction. Concentrations of FR in patient sera were expressed as dilution until absorbance reached background levels of media alone.

T cell tracking

A fraction of transduced T cell cultures (17–50%, 1.5 × 10⁹ to 7.5 × 10⁹ cells) were radiolabeled with ¹¹¹In-oxine as described previously (26). Briefly, this involved incubation with 750 μCi ¹¹¹In-oxine per 10¹⁰ cells in 30 to 50 mL PBS for 15 minutes with gentle rocking. Labeled cells were then washed and resuspended in 100 mL of saline containing 5% human serum albumin and 75,000 IU IL-2 for i.v. infusion into patients over a period of 10 to 20 minutes. Gamma camera images were obtained at intervals for up to 5 days where practicable.

Characterization of gene-modified T cells used in cohort 1

A complete characterization of T cells used in cohort 1 has already been described previously (22), but briefly, T cell cultures were stimulated with anti-CD3 antibody and were shown to expand from 11,000- to 3,000,000-fold. The mean time of culture of T cells from patients in cohort 1 was 47 days (range, 25–56 days). The T cells secreted IFN-γ specifically in response to FR and could lyse FR⁺ tumor cells. Phenotypically, the bulk lymphocyte population was composed of both CD4⁺ and CD8⁺ T cells and was shown to consist of a diverse range of clones able to secrete a variety of cytokines, including IFN-γ, IL-10, granulocyte macrophage colony-stimulating factor, and IL-2, in response to FR. Percentages of CD4⁺CD8⁻ T cells varied between 1% and 39%, and CD4⁻ CD8⁺ T cell percentages ranged from 47% to 94%.

Expansion of T cells used in cohort 2

In this cohort of the study, T cells received an initial stimulation with allogeneic stimulator cells and transduction with retroviral vector for a culture period of between 21 and 38 days. During this stimulation period, T cell expansion from patients varied from 12- to 325-fold. To further expand T cell numbers and enrich for allo-specific T cells, a second stimulation with PBMCs from the original PBMC donor was done. This restimulation resulted in a further expansion in T cells of ~50-fold. A representative growth curve for T cells over two stimulations is presented in Fig. 1A. The mean time of T cell culture for cohort 2 was 40.5 days (range, 37–48 days).

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Fig. 1

Growth and phenotype of gene-modified T cells. A, following allogeneic stimulation, 8 × 10⁷ Tcells were transduced with retroviral vector encoding the MOv-γ receptor and maintained at 1to 2 × 10⁶/mL in media containing IL-2. Transduced T cells were restimulated with allogeneic PBMCs on day 21, which resulted in further expansion of Tcells. Using this method, large numbers of dual-specificTcells could be generated. T cell expansion depicted is for patient 9. Representative of all six patients in cohort 2.The phenotype of transduced Tcells from cohort 2 of the study was determined with respect to T cell subset markers and chimeric receptor expression using specific antibodies and flow cytometry. B, although the relative proportions of CD4⁺ and CD8⁺ T cells varied between patients, the culture was made up predominantly of CD4⁺ and CD8⁺ cells as seen in the representative plot. C, expression of the chimeric MOv-γ receptor was evident following staining with anti-idiotype antibody (thick line) compared with isotype control antibody (thin line). Representative of all patients.

To promote high levels of allo stimulation, stimulator PBMCs were HLA typed at MHC class I loci to check for allelic differences to patient HLA type. Patient HLA type was determined to be largely dissimilar to stimulator HLA, with differences in at least four of six alleles.

Phenotype of T cells in cohort 2

T cells are characterized into two major phenotypic subsets, either CD4⁺ or CD8⁺, which have different fundamental abilities of helper function or cytotoxic function, respectively. Because this could affect on the overall function of bulk T cell populations and interpretation of clinical results, the relative proportions of CD4⁺ and CD8⁺ T cells were determined for each culture. Following two stimulations with allogeneic PBMCs, expression of CD4 and CD8 T cell markers was determined using specific monoclonal antibodies and flow cytometry. Percentages of CD4⁺CD8⁻ T cells varied between 2% and 82%, and CD4⁻ CD8⁺ T cell percentages ranged from 13% to 85%. CD4⁻CD8⁻ cells were present in all cultures but only as a minor population (2–15%; Fig. 1B).

All T cells were transduced, as indicated by G418 resistance, and expressed the chimeric MOv-γ receptor, as determined, in flow cytometry, by an increase in fluorescence staining in presence of Id 18.1, an anti-MOv18 idiotype monoclonal antibody compared with staining in presence of isotype control antibody (Fig. 1C). Although expressed at low level, the chimeric receptor endowed T cells with the ability to respond specifically against FR⁺ target cells (see next section).

IFN-γ secretion by T cells in response to tumor cells and allogeneic stimulator PBMC

T cell cultures from patients in cohort 1 were shown to secrete IFN-γ specifically in response to FR, and this has been previously reported (22). IFN-γ levels in response to FR⁺ IGROV-1 cells varied from 1,749 to 28,560 pg/mL. With respect to patients in cohort 2, an important requirement of dual-specific T cells was their ability to respond to both FR and allogeneic stimulator PBMCs that were to be used as immunogen following T cell transfer. IFN-γ secretion following coculture of T cells with tumor or allogeneic PBMC was used as an indicator of T cell response. Although there was some variation between patients in IFN-γ levels in response to FR⁺ IGROV-1 cells (1,295–9,050 pg/mL; Table 2), secretion was always greater than that in response to FR⁻ melanoma cells (14–54 pg/mL), thereby showing that transduced T cells could respond specifically against FR. The specificity of the response was also supported by the observed lack of IFN-γ secretion upon coculture of nontransduced T cells with IGROV-1 cells (data not shown).

Transduced T cells from all patients in cohort 2 also responded against their specific allogeneic stimulator PBMCs, although here again the level of IFN-γ secreted varied between patients (603–5,625 pg/mL; Table 2). No correlation was observed between response against FR and allo PBMCs, with the anti-FR response sometimes greater than, and sometimes less than, the allo response. However, OKT3-induced IFN-γ secretion was always greater than that induced by IGROV-1.

Patient treatment details

Because higher patient response rates have been observed in adoptive immunotherapy of melanoma when high-dose IL-2 is included in the treatment regimen, we included administration of this T cell growth factor in this cohort of the phase I study. Patients without evidence of ischemic heart disease or a history of congestive heart failure or cardiac arrhythmias or other debilitating symptoms received i.v. IL-2 at 720,000 IU/kg every 12 hours up to six doses if adequately tolerated.

The first four patients in cohort 1 received an escalating dose of T cells over three cycles beginning at 3 × 10⁹ before progressing in the next cycle (if tolerated) to 1 × 10¹⁰ and ultimately to 3 × 10¹⁰ to 5 × 10¹⁰. There was ~4 weeks between doses. Having shown no grade 3 or 4 toxicity not easily reversible in the first four patients, the remaining patients in this cohort were eligible to receive doses of T cells at the higher levels.

A variety of symptoms indicative of treatment-related toxicity were observed with grade 1 and 2 fatigue and nausea common. In addition, some patients experienced grade 3 and 4 toxicities often including hypotension and dyspnea (Table 3). Other grade 3 and 4 toxicities less frequently encountered included fatigue, leukopenia, rigors, sinus tachycardia, and diarrhea (Table 3). It is likely that these toxicities were due to the IL-2 component of the treatment regimen because these symptoms have been observed previously in melanoma patients receiving high-dose IL-2.

A total of six patients were enrolled in cohort 2 of the study, two of whom received two cycles of therapy. Doses of T cells differed between patients, varying between 4.0 × 10⁹ and 169.0 × 10⁹, with the dose given being determined by cell yield from individual cultures after two allogeneic stimulations (Table 3). Numbers of allogeneic PBMCs used for immunization also varied (although not greatly), with doses of 4.5 × 10⁹ to 7.8 × 10⁹ given per cycle. Toxicities associated with cell transfer or allogeneic immunizations were relatively mild, only reaching grade 1 or 2. Skin reaction at the site of allogeneic immunization, consisting of erythema, was the most common side effect, although nausea was also experienced by two patients (Table 3). The low-grade toxicities observed in this cohort that received cells but no IL-2 further supports the likelihood that grade 3 and 4 toxicities observed in patients in cohort 1 were due to IL-2 administration.

Disease status was followed by a computed tomography scan done after each cycle of therapy. In addition, serum CA-125 levels were determined following therapy. No reduction in the size of any tumor was observed, and disease progressed in all patients. In addition, there was no significant reduction in CA-125 levels (data not shown).

Tracking gene-modified T cells

T cell trafficking to tumor is an important requirement for antitumor activity of T cells and has been shown to correlate with response in mice and humans (27). To determine if anti-FR–transduced T cells trafficked to sites of ovarian cancer metastases, a proportion of T cells in cohort 1 were labeled with ¹¹¹In before adoptive transfer, and imaging was done at intervals for up to 5 days later. Three patients received radiolabeled T cells during a single cycle of therapy, and another two patients received radiolabeled cells during two cycles (Table 3). Radiolabeled T cells accumulated initially in lung and subsequently in liver and spleen (Fig. 2A). Interestingly, T cells persisted longer in the lungs of patients who received T cells that had been subjected to relatively prolonged culture and restimulation in vitro. Restimulation was sometimes done to generate sufficient cell numbers for treatment. Restimulation consisted of a second round of incubation with OKT3, IL-2, and irradiated allogeneic PBMCs (100:1, PBMC/T cells). Relative lung persistence was determined by comparing the ratio of lung/liver and/or spleen ¹¹¹In signal intensity ranging from 1 to 5 days after transfer. Three of three restimulated T cell transfers persisted longer in lungs compared with four of four nonrestimulated T cell transfers (P₂ = 0.03, Fisher’s exact test). A representative image following transfer of ¹¹¹In-labeled restimulated T cells is depicted in Fig. 2B. Interestingly, increased persistence of restimulated T cells in lungs seemed to be associated with dyspnea (Table 3: patient 4, cycle 2 and patient 7, cycle 1). Reproducible, specific localization of radiolabeled T cells to ovarian tumors was not observed, although some accumulation of T cells was detected in a pelvic mass of patient 4 at 48 hours after cycle 2. (Fig. 2C).

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Fig. 2

Biodistribution of radiolabeled T cells. Patients received up to 7.5 × 10^{9 111}In-labeledTcells, and imaging was done using a gamma camera at intervals followingTcell transfer. A, representative image of four transfers that were done withTcells that received a single stimulation with OKT3. B, representative image of three transfers that were done usingTcells that had received two stimulations with OKT3. Radioisotope signal was detected in lungs, liver, and spleen. Radiolabeled cells were preferentially retained in lungs of patients that received twice stimulated Tcells. C, anteroposterior image of the abdomen of patient 4 at 48 hours after receiving T cells in cycle 2 with evidence of T cell localization to a peritoneal tumor (bottom) in addition to localization to liver (top).

Persistence of gene-modified T cells following adoptive transfer

Expansion and persistence of specific T cells is important in an effective immune response against antigen, and details of this can provide insight into the response against antigen and the regulation of adoptively transferred T cell numbers. We, therefore, determined the persistence of gene-modified anti-FR T cells in the circulation of patients and expressed this as a percentage of total blood leukocytes. Adoptively transferred T cells were detectable in all patients at most times in the days immediately following cell transfer, often making up >1% of circulating lymphocytes (Table 4). However, the percentage of circulating transduced cells quickly declined and was absent or barely detectable after ~3 weeks, with the exception of patient 12, where a low percentage of transduced T cells were detected at 12 months after transfer.

We thank My Do for technical assistance and Arnold Mixon and Shawn Farid for flow cytometric assays.

Grant support: The National Health and Medical Research Council (M. Kershaw), National Breast Cancer Foundation of Australia (M. Kershaw), Bob Parker Memorial Fund (J. Westwood), and Peter MacCallum Cancer Centre Foundation (J.Westwood).