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Clinical Cancer Research 13, 1857-1867, March 15, 2007. doi: 10.1158/1078-0432.CCR-06-1905
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Long-Lived Antitumor CD8+ Lymphocytes for Adoptive Therapy Generated Using an Artificial Antigen-Presenting Cell

Marcus O. Butler1,3,5, Jeng-Shin Lee4,5, Sascha Ansén1,3,5, Donna Neuberg2, F. Stephen Hodi1,3,5, Andrew P. Murray1, Linda Drury1,3, Alla Berezovskaya1, Richard C. Mulligan4,5,6, Lee M. Nadler1,3,5 and Naoto Hirano1,3,5

Authors' Affiliations: Departments of 1 Medical Oncology and 2 Biostatistics and Computational Biology, Dana-Farber Cancer Institute, 3 Department of Medicine, Brigham and Women's Hospital, 4 Department of Genetics, 5 Harvard Medical School, and 6 Division of Molecular Medicine, Children's Hospital, Boston, Massachusetts

Requests for reprints: Marcus Butler, Dana-Farber Cancer Institute, Department of Medical Oncology, 44 Binney Street, Boston, MA 02115. Phone: 617-632-4589; Fax: 617-632-2255; E-mail: Marcus_Butler{at}dfci.harvard.edu.


   Abstract
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Materials and Methods
 Results
 Discussion
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Purpose: Antitumor lymphocytes can be generated ex vivo unencumbered by immunoregulation found in vivo. Adoptive transfer of these cells is a promising therapeutic modality that could establish long-term antitumor immunity. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we sought to establish a clinical grade culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL).

Experimental Design: We created an off-the-shelf, standardized, and renewable artificial antigen-presenting cell (aAPC) line that coexpresses HLA class I, CD54, CD58, CD80, and the dendritic cell maturation marker CD83. We tested the ability of aAPC to generate tumor antigen-specific CTL under optimal culture conditions. The number, phenotype, effector function, and in vitro longevity of generated CTL were determined.

Results: Stimulation of CD8+ T cells with peptide-pulsed aAPC generated large numbers of functional CTL that recognized a variety of tumor antigens. These CTLs, which possess a phenotype consistent with in vivo persistence, survived ex vivo for prolonged periods of time. Clinical grade aAPC33, produced under current Good Manufacturing Practices guidelines, generated sufficient numbers of CTL within a short period of time. These CTL specifically lysed a variety of melanoma tumor lines naturally expressing a target melanoma antigen. Furthermore, antitumor CTL were easily generated in all melanoma patients examined.

Conclusions: With clinical grade aAPC33 in hand, we are now poised for clinical translation of ex vivo generated antitumor CTL for adoptive cell transfer.

The complexity of inducing clinically significant antitumor-specific immunity following in vivo vaccination has led several groups to explore the potential of adoptive immunotherapy. This modality is attractive because it facilitates the delivery of essential signals to highly selected populations of responder T cells without the confounding immunoregulatory issues that exist in the tumor-bearing host. Recent clinical successes associated with adoptive transfer of antitumor reactive T cells support the potential of this modality to have significant clinical impact. For example, adoptive transfer of donor leukocytes to patients who have relapsed following allogeneic stem cell transplantation has resulted in dramatic complete remissions and sometimes cure (1). Adoptive therapy of T cell clones directed against a melanoma-specific antigen has shown clinical activity (2). Demonstration that adoptive transfer of anti-Epstein Barr virus (EBV)-specific T cell lines can induce responses in patients with Hodgkin's disease and nasopharyngeal carcinoma is similarly compelling (3–5). Finally, significant clinical responses have been achieved by the treatment of lymphodepleted patients with adoptively transferred ex vivo activated melanoma-infiltrating lymphocytes and subsequent high-dose interleukin 2 (IL-2; ref. 6). Taken together, these encouraging clinical results suggest that if a standardized method could be developed to generate large numbers of functional antitumor-specific T cells ex vivo, then this modality might become important for the treatment of cancer patients.

The generation of antitumor-specific T cells ex vivo for adoptive T cell therapy requires the selection of an appropriate tumor rejection antigen, a means to present that antigen, and conditions supportive for the generation of antitumor-specific T cells that are both long-lived and functional. With the large numbers of potential tumor rejection antigens that have been characterized, the choice of the antigen is not likely to be a major obstacle (7, 8). In contrast, the choice of an optimal antigen-presenting cell (APC) seems to be much more challenging. The difficulties in selecting an APC capable of priming and expanding antitumor-specific T cells coupled with technical difficulties in their isolation, enrichment, expansion, and cryopreservation present considerable obstacles to the clinical investigator. Patient-derived autologous APC include numerous dendritic cell (DC) subtypes, EBV-transformed B cells, and CD40-activated B cells (9–13). Although each of these APCs has significant attributes, we and others have not been able to standardize them for adoptive therapy, and preparation of patient-specific APC is limited by regulatory complexities as well as cost.

To overcome these obstacles, a number of laboratories have sought to prepare artificial APCs (14). Artificial APCs are standardized, "off-the-shelf" reagents that are engineered to deliver the appropriate signals to generate the desired T cells. Investigators have employed allogeneic and xenogeneic cell lines as well as polystyrene beads as APC platforms to generate and expand antigen-specific T cells ex vivo (15–19). Although capable of generating antitumor-specific T cells, these artificial APCs are encumbered by factors that negatively impact their translation to widespread clinical practice. In the case of nonhuman cell lines (15–17), xenogeneic MHC and antigens might lead to ineffective immunity, which could dominate over desired antitumor-specific immune response. Requirements for allogeneic feeder cells and ill-defined supernatants in polystyrene bead APC systems will likely lead to regulatory scrutiny before widespread use (18). The allogeneic artificial APC reported by Maus et al. (19) required tetramer-guided cell sorting under current Good Manufacturing Practices (cGMP) conditions, which poses a significant obstacle to most other clinical centers. Because the above issues might limit translation of an artificial APC, we sought to develop an artificial APC and culture conditions that would overcome many of these obstacles.

We have attempted to identify the characteristics that would define an optimal artificial APC to generate human CD8+ T cells for adoptive T cell therapy. For the APC itself, it should be (a) based on a human cell line backbone that does not express MHC or negative signals, (b) off the shelf, (c) capable of self-renewal, and (d) deliver all the appropriate signals necessary for CD8+ T cell activation and function. Antitumor-specific CD8+ T cells generated by this APC must be capable of (a) priming to a specific antigen ex vivo, (b) expanding to sufficient numbers ex vivo for adoptive T cell therapy, (c) exhibiting potent effector function including cytotoxicity and IFN-{gamma} production, (d) surviving ex vivo for prolonged periods of time for repetitive treatment, (e) localizing in vivo to sites of tumor, and (f) persisting for prolonged periods of time in vivo as memory T cells. In this study, we have developed the artificial APC (aAPC) that meets all of the above criteria. The antitumor-specific T cells generated by aAPC meet all except the in vivo criteria. A clinical trial is necessary to examine the outstanding issues of localization and memory.


   Materials and Methods
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Phenotypic analysis. Flow cytometry analysis was done using the following fluorochrome-conjugated monoclonal antibodies (mAbs): anti-human CD40, CD54, CD58, CD80, CD86, CD70, CD95, CD206, CD209, B7-H2, CD40 ligand, 4-1BB ligand, HLA-ABC, and HLA-DR, DP, DQ (BD PharMingen, San Diego, CA); CD8, CD25, and CD28 (Beckman-Coulter, Miami, FL); CD16, CD27, CD32, CD45RA, CD45RO, CD62L, CD64, and CD83 (Caltag, Burlingame, CA); and CCR7 (R&D Systems, Minneapolis, MN). Biotinylated antihuman PD-L2, B7-H1, and B7-H3 goat antibodies were used (R&D Systems). Vß staining of antigen-specific T cells was done using the Beta Mark TCR Vbeta Repertoire kit (Beckman-Coulter). Isotype control antibodies were obtained from BD PharMingen. Biotinylated control goat antibodies were purchased from Jackson Immunoresearch (West Grove, PA). In the case of cytotoxic T lymphocyte (CTL) phenotype analysis, tetramer staining was done immediately before staining for surface antigens.

ELISA detection of cytokines in K562 supernatants. Cytokine expression profile was determined by protein ELISA. One million K562 cells were incubated in complete media for 24 h at 37°C. Supernatants were collected, centrifuged to remove cellular debris, and stored at –20°C. ELISA analysis was done per manufacturer's recommendations (R&D Systems; except IL-7, Cell Sciences, Canton, MA). Standard curves for macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), MIP-1ß regulated upon activation, normal T cell expressed and secreted, and IL-2 sR{alpha} were extended to include lower concentrations.

Generation of aAPC, clinical grade aAPC33, and mature DC. To generate aAPC, K562 (American Type Culture Collection, Manassas, VA) was transduced using a highly efficient retroviral vector as described previously (20). Briefly, cDNAs encoding HLA-A*0201 (A2), CD80, and CD83 were subcloned into the retroviral vector pMX. The vectors were transfected into a 293GPG packaging cell line (21) and replication-defective virus supernatants were harvested. After infection of K562 cells with each supernatant, antibody-directed flow cytometry sorting was done to obtain high expression for all three genes.

To avoid exposure to retrovirus, clinical grade aAPC33 was constructed by plasmid transfection. cDNAs encoding HLA-A2, CD80, and CD83 were subcloned into the cytomegalovirus promoter–driven expression plasmid pUC-MD.7 K562 was simultaneously transfected with these expression plasmids and pJ6Omega-puro, which encodes a puromycin resistance gene, by lipofection (X-tremeGene Q2, Roche, Indianapolis, IN). Seventy-one clones, established by limiting dilution after puromycin selection (2 µg/mL), were assessed for expression of HLA-A2, CD80, and CD83. High-expression clones were analyzed for their ability to stimulate MART1/Melan-A–specific T cells. aAPC33 was found to consistently generate the highest number of antigen-specific T cells in 3/3 donors and had sufficient growth characteristics. A master cell bank and clinical lots were established and have undergone extensive quality control testing. All experiments with aAPC33 were done using clinical lots.

DC were generated from purified monocytes using IL-4 (10 ng/mL, PeproTech, Rocky Hill, NJ) and granulocyte macrophage colony-stimulating factor (GM-CSF, 50 ng/mL; Immunex, Seattle, WA) in RPMI 1640 (Mediatech) supplemented with 10% FCS (Invitrogen, Carlsbad, CA). Maturation of DC was induced by dsRNA (25 µg/mL; Sigma, St. Louis, MO) and tumor necrosis factor-{alpha} (TNF-{alpha}, 50 ng/mL; PeproTech; ref. 22).

Generation of antigen-specific CTL. HLA-A2–positive peripheral blood mononuclear cells (PBMC) were obtained by apheresis of healthy donors or peripheral blood draw from melanoma patients. Appropriate informed consent and institutional review board approval were obtained. Patients 1 and 3 had a history of locally advanced melanoma, whereas patient 2 had widely metastatic melanoma to the skin, lung, adrenal glands, and lymph nodes at the time of phlebotomy. To establish antigen-specific T cell lines, purified CD8+ T cells were obtained by positive selection (Dynal, Oslo, Norway or Miltenyi Biotec, Gladbach, Germany). APCs were pulsed with peptide at 10 µg/mL in serum-free RPMI for 8 to 10 h at room temperature. Peptides used were MART1/Melan-A (AAGIGILTV), NY-ESO-1 (SLLMWITQC), telomerase (ILAKFLHWL), Her-2/neu (KIFGSLAFL), influenza matrix protein (GILGFVFTL), and HIV pol (ILKEPVHGV). Generation of MART1/Melan-A T cells with aAPC33 and matured DC was done with the heteroclitic peptide (ELAGIGILTV). APCs were then radiated with 20,000 rads (aAPC) or 3,000 rads (PBMC or DC), washed, and added to purified CD8+ T cells at a ratio of 1:20 in 24-well plates in RPMI supplemented with gentamicin (50 µg/mL) and 10% human AB sera (Nabi). Peptide-pulsed PBMC were added to CD8+ T cells at a ratio of 1:5 where indicated. For large-scale cultures, cells were incubated in gas-permeable VueLife bags instead of plates (American Fluoroseal Corporation, Gaithersburg, MD). Beginning the next day, CTL cultures were supplemented with IL-2 (Chiron, Emeryville, CA) and IL-15 (Peprotech) every 3 to 4 days as indicated. Repeat stimulations were done every 7 to 14 days as indicated. Following multiple rounds of stimulation, lines were evaluated for tetramer staining, cytotoxicity, and/or IFN-{gamma} secretion. The percentage of tetramer-stained cells and the number of viable T cells were used to determine the total numbers of generated antigen-specific CTL.

Tetramer assays, cytotoxicity and IFN-{gamma} ELISPOT assays. Tetramer analysis was done as described previously (23). Cytotoxicity and ELISPOT assays were done as previously described (24, 25). Peptides used for these assays are identical to those used for tetramer assays with the exception of MART1/Melan-A (AAGIGILTV).

Statistical analysis. Data were analyzed as the ratio of CD8+ or MART1/Melan-A tetramer-staining T cells treated with IL-2 and IL-15 compared with the number of T cells treated with IL-2 alone for each sample. The Student's t test was used to assess whether the ratio was >1.0. All statistical testings were conducted at the two-sided 0.05 significance level.


   Results
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 Results
Discussion
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Artificial APCs using a K562 backbone support the generation of antigen-specific CD8+ T cells. K562 displays characteristics that make it an ideal platform for generating an APC line (Tables 1 and 2 ). Importantly, K562 does not express any HLA class I or class II molecules on its cell surface (19, 26, 27). Also, other immunologically relevant molecules are largely absent. We did not detect protein expression of ICOS-L, PD-L1, PD-L2, CD40, CD40 ligand, DC-Sign, CD27, CD70, CD83, CD86, or 4-1BB ligand on the cell surface. Additionally, whereas CD32 expression is positive, neither the mannose receptor nor other Fc{gamma} receptors is expressed (Table 1). Furthermore, it does not secrete IL-2, IL-7, IL-10, IL-15, IFN-{gamma}, or GM-CSF as determined by ELISA (Table 2). In addition, we showed the absence of the T cell growth factors IL-2, IL-4, IL-15, and IL-21 by performing the murine CTLL-2 cell line–based biological assay (data not shown).


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  		Table 1. Cell surface expression of immunoaccessory molecules on K562 examined by flow cytometry

 

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  		Table 2. Cytokine secretion by K562 analyzed by ELISA

 
Although relatively null, K562 does express several molecules that may enhance its ability to serve as an APC. The adhesion molecules, CD54 and CD58, which are important in generating efficient T cell responses are highly expressed. Additionally, the B7 family members, CD80 (weakly) and B7-H3, are present on the cell surface of K562 (Table 1). Interestingly, K562 was also found to secrete measurable levels of inflammatory cytokines such as IL-6, IL-6sR, IL-8, MCP-1, MIP-1{alpha}, and regulated upon activation, normal T cell expressed and secreted, which are involved in the priming, growth, and chemoattraction of T cells. K562 also secretes the inhibitory cytokine, transforming growth factor-ß1 (TGF-ß1), although in a range moderately above baseline levels in media tested (Table 2).

Considering these properties, we developed aAPC by first engineering K562 to express the widely shared HLA class I molecule, HLA-A*0201, and then engineering the expression of the immunostimulatory molecules CD80 and CD83. We have previously shown that CD83 induces preferential enrichment and prolonged expansion of antigen-specific CD8+ T cells in a CD80-dependent manner (23). High-level expression was achieved by flow cytometry cell sorting and was stable for more than 2 months in culture (Fig. 1A ).


Figure 1
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  		Fig. 1. aAPC supports the generation of antigen specific CD8+ T cells. A, expression of transduced A2, CD80, and CD83 on aAPC was determined by flow cytometry with specific antibody (white) and isotype control (black). B, aAPC prime and expand antigen-specific CD8+ T cells. Positively selected CD8+ T cells from normal A2+ donors were stimulated with MART1/Melan-A peptide-pulsed aAPC on a weekly basis. Cultures were supplemented with IL-2 (10 IU/mL) between stimulations. Generated T cell lines were stained with MART1/Melan-A tetramer followed by anti-CD8 mAb after the third stimulation. Representative tetramer staining representing four separate donors is shown.

 
We then showed that peptide-pulsed aAPC can efficiently support the expansion of antigen-specific T cells. CD8+ T cells were obtained by positive selection from normal donors and stimulated once a week with aAPC pulsed with MART1/Melan-A peptide. Cultures were supplemented with IL-2 every 3 to 4 days between stimulations. One week following the third stimulation, CTL lines were found to display antigen-specific T cells when stained with MART1/Melan-A tetramer (Fig. 1B). Additionally, the CTL lines showed potent cytoxicity using peptide-pulsed T2 cells in a standard chromium release assay (Fig. 2A ).


Figure 2
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  		Fig. 2. IL-15 promotes the acquisition of antigen-specific IFN-{gamma} secretion and antigen-specific T cell expansion. Analysis of aAPC-stimulated T cell cultures that were supplemented with either IL-2 (10 IU/mL) and/or IL-15 (10 ng/mL) was done following the third stimulation. A, incubation with IL-15, but not IL-2, promotes the acquisition of antigen-specific IFN-{gamma} secretion by MART1/Melan-A–specific T cells primed ex vivo. Tetramer staining was done as described and showed the generation of antigen-specific T cells. IFN-{gamma} ELISPOT was done by incubating T cells with T2 cells in the presence of 10 µg/mL MART1/Melan-A peptide. When cultures were supplemented with IL-2, no antigen-specific IFN-{gamma} secretion was observed. In contrast, when cultures were supplemented with IL-15, antigen-specific IFN-{gamma} secretion was acquired. Standard cytotoxicity assay was done using peptide-pulsed T2 cells as targets ({blacksquare}, MART1/Melan-A peptide; or bullet, human T-cell lymphotrophic virus-1 Tax peptide as control). Assays representing three separate donors are shown. B, incubation with a combination of IL-2 and IL-15 increases the number of CD8+ and MART1/Melan-A–specific T cells. A2+ CD8+ T cells from normal donors were stimulated with MART1/Melan-A peptide-pulsed aAPC thrice on a weekly basis. Between stimulations, cultures were either supplemented with IL-2 alone (10 IU/mL) or a combination of IL-2 (10 IU/mL) and IL-15 (10 ng/mL). After three rounds of simulation, cultures were stained with relevant tetramer to determine the percentage of MART1/Melan-A–specific T cells. Significantly, more CD8+ (P = 0.02) and MART1/Melan-A–specific (P = 0.03) T cells were generated when cultures were supplemented with a combination of IL-2 and IL-15. C and D, expansion of antigen-specific T cells is a consequence of both increased proliferation and decreased apoptosis. Representative assays from two separate donors are shown. C, proliferating fraction is shown as a percentage of MART1/Melan-A tetramer staining T cells incorporating BrdUrd. D, fraction of cells undergoing apoptosis is shown as a percentage of MART1/Melan-A tetramer staining T cells staining annexin V.

 
Coculture with IL-15 promotes the acquisition of IFN-{gamma} secretion and enhances antigen-specific T-cell expansion. We were also able to generate flu-specific T cells using aAPC pulsed with the influenza (flu) matrix protein-derived peptide (GILGFVFTL). The T cells were able to induce cytotoxicity when tested in a standard chromium release assay. Additionally, flu-specific T cells were able to secrete IFN-{gamma} as measured by ELISPOT (data not shown). In contrast, when we studied MART1/Melan-A responses in normal/naïve donors, antigen-specific T cells did not secrete IFN-{gamma} in response to antigen, suggesting that additional signals are required for antigen-inexperienced T cells to acquire this function when stimulated in vitro using aAPC. Therefore, we investigated whether IL-15, a cytokine secreted by DC and implicated in the homeostatic proliferation and differentiation of CD8+ T cells, might enhance MART1/Melan-A–specific T cell effector function. We generated MART1/Melan-A–specific T cells using experimental conditions used in Fig. 1B, except that cultures were supplemented with either IL-2 or IL-15. Seven days following the third stimulation, both conditions produced antigen-specific CTL detected by tetramer staining. However, despite the fact that the percentage of tetramer staining MART1/Melan-A T cells is higher when generated with IL-2, ELISPOT detected virtually no antigen-specific IFN-{gamma} secretion. On the other hand, antigen-specific IFN-{gamma} secretion was easily shown in cultures that were supplemented with IL-15 (Fig. 2A). Both cultures acquired potent cytotoxicity function, and differences in the magnitude can be accounted for by the percentage of antigen-specific T cells present (Fig. 2A).

We next investigated the effect of IL-15 on the number of generated antigen-specific T cells. The addition of both IL-2 and IL-15 to T cell cultures increased the total number of MART1/Melan-A tetramer-staining T cells (Fig. 2B, right) in every donor tested by a mean of 2.0-fold (range, 1.4-2.65; P = 0.03). These CTL cultures displayed potent effector function as measured by the cytotoxicity assay and IL-2 and IFN-{gamma} ELISPOT assays (data not shown). The combination of IL-2 and IL-15 in the T cell cultures resulted in increased proliferation as measured by bromodeoxyuridine (BrdUrd) incorporation (Fig. 2C). Additionally, annexin V positivity was decreased, indicating that apoptosis was inhibited (Fig. 2D). Similar results were observed when flu-specific CTL were generated (data not shown). Therefore, optimal conditions to generate antigen-specific CTL with peptide-pulsed aAPC include supplementing cultures with a combination of IL-2 and IL-15.

Using these conditions, we compared the ability of aAPC and PBMC to generate CTL in three healthy donors. After three weekly stimulations, CTL lines were harvested and analyzed for MART1/Melan-A specificity. In each donor tested, more MART1/Melan-A CTL were generated with aAPC compared with CTL lines stimulated with PBMC by an average of 127-fold (range, 89-189).

Long-lived effector memory CTL can be generated with aAPC against multiple antigens. Our artificial APC-based system satisfies the requirement of generating antigen-specific T cells against many antigens in multiple donors. As shown in Table 3 , our laboratory has been able to generate antigen-specific T cell lines to Her-2/neu, NY-ESO-1, telomerase, and MART-1 in multiple donors. Therefore, this T cell generation strategy could be useful in the treatment of a wide range of malignancies.


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  		Table 3. Establishment of antigen-specific CTL lines

 
Also shown in Table 3, in some donors, we maintained T cell lines for remarkably prolonged culture periods, more than a year in some instances. The cell lines retained antigen specificity without T cell selection by tetramer-guided sorting or cloning by limited dilution. For example, one NY-ESO-1–specific T cell line that was maintained in culture for more than 1.5 years (Fig. 3A ) retained antigenic specificity and effector function as shown by tetramer staining (3-10%) and killing of peptide-pulsed targets (Fig. 3B and C). IFN-{gamma} secretion, however, was significantly diminished following several months in culture. Although antigen-specific IFN-{gamma} secretion was observed following 1 year of culture, <5% of the tetramer-staining cells were shown to secrete IFN-{gamma} by ELISPOT, consistent with a decrease in cytokine secretion ability following long-term cell culture (data not shown). These long-lived T cells do not seem to be terminally differentiated as indicated by the lack of CD45RA expression and are CCR7– and CD62L–, which is consistent with the proposed "effector memory" T cell phenotype (Fig. 3D). Additionally, TCR Vß analysis of the cells staining with NY-ESO-1 tetramer revealed that tetramer-positive cells were predominantly Vß 17 positive, indicating that long-term cultures consisted of a single clone (Fig. 3E). As shown in Fig. 3F, long-lived CTL are capable of killing tumor targets endogenously expressing NY-ESO-1 protein in an antigen-specific manner.


Figure 3
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  		Fig. 3. aAPC supports the long-term maintenance of antigen-specific T cells in vitro. An example of the long-term maintenance of an antigen-specific T cell line is shown. NY-ESO-1–specific T cells were maintained in vitro for >1.5 yrs. A2+ CD8+ T cells were maintained by stimulation with peptide-pulsed aAPC once every 7 to 14 d, and cultures were supplemented with IL-2 (5-10 IU/mL) and IL-15 (5-10 ng/mL). A, expansion of the total number of CD8+ T cells. T cells remained viable over the period shown. B, NY-ESO-1 tetramer staining showed the antigen specificity of long-lived CD8 T cells at day 616. C, T cell lines retained potent antigen-specific effector function as measured by cytotoxicity. Cytotoxicity assay was done using peptide-pulsed T2 cells as targets ({blacksquare}, NY-ESO-1 peptide; or bullet, HIV pol peptide as control). D, surface phenotype of NY-ESO-1–specific T cells maintained in culture for >1.5 yrs. The expression of each molecule on tetramer+/CD8+ T cells is shown. Open curves, staining for indicated molecules; shaded curves, isotype controls. E, T cells were costained with NY-ESO-1–specific tetramers and mAbs for TCR Vß subtypes. Percentage of NY-ESO-1–specific T cells expressing each subtype is shown. F, cytotoxicity assay was done using HLA-A2+ melanoma cell lines pretreated with IFN-{gamma} ({blacksquare}, A375, NY-ESO-1+; or bullet, control SK-Mel-21, NY-ESO-1–).

 
Clinical grade aAPC33 is equivalent to mature DC in the generation of large numbers of MART1/Melan-A–specific CTL. We have produced an aAPC line under cGMP conditions to apply our system clinically. Given recent concerns regarding the therapeutic use of retroviruses, a clinical version of aAPC was produced by transfecting K562 simultaneously with nonretroviral plasmids encoding the HLA-A2, CD80, CD83, and a puromycin resistance gene. To obtain a standardized aAPC, drug selection and cloning by limiting dilution were done. An aAPC clone, aAPC33, was selected that stably expressed high levels of the transfected molecules and consistently induced expansion of MART1/Melan-A peptide-specific T cells. Cultures of cGMP grade clinical lots confirmed the stable expression of transfected molecules for more than 60 days (data not shown). We then did preclinical experiments to show that peptide-pulsed aAPC33 generated large numbers of antigen-specific T cells (Fig. 4A ). In these experiments, T cell lines from healthy donors were enriched for MART1/Melan-A T cells with tetramer staining ranging between 10% and 66%. Adoptive transfer of these cells should have a favorable toxicity profile because CTL lines kill the MART1/Melan-A–expressing melanoma line, Malme-3M, but not Malme-3, a MART1/Melan-A–negative, fibroblast cell line derived from the same individual (Fig. 4B). Furthermore, CTL lines have TCR avidity high enough to kill multiple A2+ melanoma lines that express MART1/Melan-A (Fig. 4C).


Figure 4
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  		Fig. 4. Peptide-pulsed dendritic cells and aAPC33 show similar efficiency in generating MART1/Melan-A–specific T cells in vitro. Six million A2+ CD8+ T cells from normal donors were stimulated with aAPC33 pulsed with the heteroclitic MART1/Melan-A peptide once a week, and cultures were supplemented with IL-2 (10-50 IU/mL) and IL-15 (10-50 ng/mL) between stimulations. A, total number of MART1/Melan-A–specific T cells was calculated by determining the percentage of tetramer-staining T cells and the number of T cells present at the indicated times. Rapid expansion was observed in all five donors tested. B, antigen specificity was confirmed by the standard cytotoxicity assay. MART1/Melan-A–specific T cells specifically killed the tumor line, Malme-3M, which is positive for A2 and expresses the MART1/Melan-A antigen. T cells were unable to kill the fibroblast cell line Malme-3, which was derived from the same A2+ donor and does not express MART1/Melan-A. C, the ability to target endogenously expressed antigen was confirmed by the standard cytotoxicity assay. Antigen-specific cytotoxicity of A2+ MART1/Melan-A expressing lines C32, Malme-3M, Me276, and WM266-4 was shown compared with the A2+ MART1/Melan-A–negative line A375. D, T cells were costained with MART1/Melan-A–specific tetramers and mAbs for TCR Vß subtypes. Percentage of MART1/Melan-A–specific T cells expressing each subtype is shown. E, aAPC is able to stimulate MART1/Melan-A–specific T cell responses with comparable efficiency to matured DC. A2+ CD8+ T cells from two donors were stimulated by MART1/Melan-A–pulsed APC on a weekly basis, and cultures were supplemented with low-dose IL-2 and IL-15. Total number of generated MART1/Melan-A T cells was determined by tetramer staining. Surface phenotype (F) and intracellular staining (G) of MART1/Melan-A–specific T cells generated from a healthy A2+ donor are shown. The expression of each molecule on tetramer+/CD8+ T cells is shown. H, MART1/Melan-A–specific CTL lines generated from two healthy A2+ donors were stained for CD25 and Foxp3. CD25+ cells lacked expression of Foxp3 by intracellular staining. Open curves, staining for indicated molecules; shaded curves, isotype controls.

 
Next, we compared the ability of aAPC33 and mature DC to generate MART1/Melan-A–specific T cells. Immature DC were generated from peripheral blood monocytes in the presence of GM-CSF and IL-4. To obtain mature DC, we used TNF-{alpha} and double-stranded RNA, a combination which, in comparative studies, has been shown to induce superior complete activation of DC (22). The use of prostaglandin E2 was avoided because it has recently been shown that prostaglandin E2 can induce the expression of indoleamine 2,3-dioxygenase, resulting in the generation of tolerogenic DC (28). Purified A2+ CD8+ T cells were stimulated with MART1/Melan-A peptide-pulsed APC (DC or aAPC33) on a weekly basis and analyzed for tetramer staining. As shown in Fig. 4E, the generation of MART1/Melan-A–specific T cells with aAPC33 was comparable to mature DC. Detailed phenotypic analysis of MART1/Melan-A–specific T cells is shown in Fig. 4F and G. No Foxp3 staining was shown in CD25-positive cells (Fig. 4H).

Emergence of MART1/Melan-A–specific CTL with an effector phenotype from melanoma patients generated with aAPC33. To test the feasibility of generating large-scale MART1/Melan-A–specific CTL, 0.21 x 109 CD8+ T cells were positively selected from 109 PBMC that were obtained by leukopheresis of a healthy donor. Three stimulations with MART1/Melan-A–pulsed aAPC33 were done as described above except that cultures were expanded in gas-permeable bags. Following 3 weeks of culture, a total of 2.0 x 109 cells were generated, of which 32% stained with the MART1/Melan-A tetramer (Fig. 5A ). This represents an expansion of MART1/Melan-A–specific T cells by more than 4,000-fold, consistent with expansion rates observed in cultures done in 24-well plates. Phenotypic analysis showed mixed staining for CD62L and CCR7, suggesting a polyclonal status with cells expressing the proposed "central memory" phenotype (CD45RA– CD62L+ CCR7+) or the proposed effector memory phenotype (CD45RA– CD62L– CCR7–). With additional rounds of stimulation, antigen-specific cells progressively lost CD62L and CCR7 while retaining CD27 expression consistent with the emergence of an effector memory phenotype (Fig. 5A).


Figure 5
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  		Fig. 5. Effector memory phenotype emerges with repeated stimulation of healthy donor and melanoma patient T cells. A2+ CD8+ T cells were stimulated with aAPC33 pulsed with the heteroclitic MART1/Melan-A peptide once a week, and cultures were supplemented with IL-2 (10-50 IU/mL) and IL-15 (10-50 ng/mL) between stimulations. A, surface phenotype of MART1/Melan-A–specific T cells generated in large-scale CTL culture from a healthy A2+ donor at different time points in culture. The expression of each molecule on tetramer+/CD8+ T cells is shown. Open curves, staining for indicated molecules; shaded curves, represent isotype controls. B, tetramer staining of CTL cultures from three melanoma patients. CD8+ T cells from 30 to 40 mL of blood were stimulated thrice on a weekly basis. About 10 to 18 million total cells with 18% to 49% MART1/Melan-A–specific CTL were generated. C, surface phenotype of MART1/Melan-A–specific T cells. Open curves, staining for indicated molecules; shaded curves, represent isotype controls.

 
We also tested the use of aAPC33 in the generation of CTL from melanoma patients. As shown in Fig. 5B, CTL lines with 18% to 49% MART1/Melan-A–specific T cells were generated from peripheral blood draws within 3 weeks, representing an expansion of MART1/Melan-A–specific cells by more than 2 to 3 logs. Analysis of effector function confirmed specific cytotoxicity of the A2+, MART1/Melan-A+ melanoma cell line Malme-3M, but not the A2+, MART1/Melan-A– fibroblast cell line Malme-3 (data not shown). Phenotypic analysis of tetramer-stained cells (Fig. 5C) showed that a majority of cells are CD45RA– CD62L– CCR7–, consistent with the proposed effector memory T cell phenotype.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this report, we show that aAPC can be used to generate large numbers of long-lived and functionally competent antitumor-specific CD8+ T cells for adoptive immunotherapy. aAPC fulfills our proposed criteria for an artificial APC because it is a stably engineered cell line and can serve as a standardized, renewable, and unlimited source of APCs. One virtue of using this particular artificial APC system is that the parental human cell line, K562, does not express either allo- or xenogeneic MHC and is relatively free of immunoinhibitory molecules. Because engineered aAPC coexpress HLA-A2, CD80, and CD83 as well as endogenously express both CD54 and CD58, these APCs, pulsed with a variety of tumor-associated peptide antigens, provide the major signals necessary to generate antigen-specific CD8+ T cell lines. To translate these findings to clinical practice, we have successfully manufactured a master cell bank and clinical lots of aAPC33 in a cGMP facility.

aAPC provides a robust platform for the generation of large numbers of functional antigen-specific CD8+ T cells. In addition to HLA class I, CD54, CD58, and CD80, our aAPC also expresses CD83, which is highly expressed by professional APC such as mature DC, and is implicated in the development of T cell immunity by its presence in the interfollicular T cell–rich regions of the lymph node (29–31). We have previously shown that CD83 ligand is induced on activated CD4+ and CD8+ T cells, and that CD83L signaling enhances the expansion of antigen-specific CD8+ T cells (23). Coimmobilized soluble CD83 and anti-CD3 enhances CD8+ T cell expansion, and CD83 expression by tumor lines may facilitate the in vitro generation of CTL (32). In vivo vaccination with CD83-transduced tumor cell lines induces enhanced antitumor immunity, and injection of CD83-Ig fusion protein can increase the outgrowth of transplanted tumor cells and decrease tumor-directed CTL responses (32, 33). We have also found that the addition of IL-15 to our culture conditions improves the generation of functional T cells. IL-15, which has been shown to be important for the proliferation and survival of naïve and memory T cells (34), enhanced the ability of our aAPC to expand antigen-specific T cells by both inhibiting apoptosis and stimulating proliferation. Furthermore, we found that IL-15 promotes the acquisition of IFN-{gamma} secretion by ex vivo primed MART1/Melan-A–specific T cells. This is consistent with previous reports that IL-15 contributes to the acquisition of effector function of naïve T cells (35), and that T cells cultured with IL-15 can have much greater antitumor activity in models of adoptive T cell transfer (36, 37). Our data suggest that IL-15, but not IL-2, can directly modulate the ability of newly primed T cells to secrete IFN-{gamma}. Although IL-2 and IL-15 signal through Janus-activated kinase-1 and Janus-activated kinase-3 via the common ß and {gamma} chains, IL-15 alone binds to the IL-15 {alpha} chain, which signals through TRAF2 (38). Therefore, it is conceivable that TRAF2 mediates the IL-15 signal to T-bet/TBX21 and/or EOMES and enables antigen-specific IFN-{gamma} secretion (39). Because the capacity to secrete IFN-{gamma} is associated with antitumor activity of adoptively transferred T cells, we have incorporated IL-15 into our culture conditions.

We have shown that clinical grade aAPC33 performed comparably to mature DC in generating CTL specific for MART1/Melan-A, an antigen for which there is a high precursor CTL frequency (40). Within 3 to 4 weeks, we were able to generate substantial numbers of MART1/Melan-A–specific T cells in healthy donors and melanoma patients, including those with widely metastatic disease (Figs. 4A and 5A and B). Given this, we predict that in the majority of patients, it will be feasible to infuse between 2 x 108/m2 and 2 x 109/m2 total cells within 3 weeks following a single leukopheresis. Although the immunogenicity of aAPC33 was comparable to that of mature DC for MART1/Melan-A (Fig. 4C), we are concerned that the in vitro generation of CTL with aAPC might be more difficult for other antigens in which the precursor CTL frequency is low. For such antigens, in vivo vaccination might increase the frequency of antigen-specific CTL with high avidity and therefore make clinically relevant ex vivo expansion feasible.

A clinical trial of adoptive transfer in humans will be required to definitively determine whether aAPC-generated CTL can persist in vivo. However, in vitro evidence suggests that stimulation with aAPC does not induce exhaustion of CTL. Functional aAPC-generated CTL lines could be maintained in culture for a prolonged period of time, in some cases for more than a year (Fig. 3 and Table 3). Our long-term cultured T cells were not terminally differentiated and continued to display an effector memory phenotype (CD45RA– CD62L– CCR7–; ref. 41). Importantly, these cells were CD27+ CD28+, which has been associated with the long-term persistence of adoptively transferred tumor-infiltrating lymphocytes and CTL clones in humans (42, 43). It is likely that two factors contributing to the longevity of these cultures include the expression of CD83 by stimulating aAPC and the addition of IL-15 to cultures. Engagement of CD83 with CD83 ligand on antigen-specific T cells results in an increase in proliferation and an inhibition of apoptosis (23). Additionally, IL-15 has been shown to contribute to T cell survival resulting in enhanced antitumor activity in vivo (36, 37).

These long-lived T cell lines could provide a supply of CTL for multiple adoptive T cell infusions in the longitudinal treatment of cancer patients. However, the potential for in vivo expansion might be higher for T cells that have undergone fewer cell divisions ex vivo. As shown in Fig. 5A, early CTL cultures generated with this system have a higher percentage of antigen-specific cells that express the proposed central memory phenotype (CCR7+ and CD62L+). These cells might more readily respond to antigenic stimulation given their capacity to home to the lymph node, where they may be induced to expand and generate both memory and antitumor effector cells (41). Also, as shown in Fig. 4D, TCR Vß chain usage analysis of tetramer-positive cells revealed that antigen-specific T cells in these cultures are polyclonal, suggesting the presence of antigen-specific T cells with different avidities and phenotypes. Consequently, the most fit antitumor CTL could be selected in vivo following adoptive transfer. In murine tumor models, prolonged culture of T cells decreases their effectiveness in eradicating tumors (44, 45). This is also in agreement with recent observations in the adoptive transfer of ex vivo activated tumor-infiltrating lymphocytes (6, 46). We have also observed that, whereas long-term cultured T cells retain cytotoxicity, antigen-specific IFN-{gamma} secretion declined after multiple rounds of stimulation. Furthermore, it has been shown that the telomere length of transferred lymphocytes correlates with in vivo persistence and tumor regression in melanoma patients receiving cell transfer therapy (46). Taken all together, infusion of polyclonal, younger T cells generated in our cultures might be more clinically efficacious than that of older T cells cultured for prolonged time. With an unlimited supply of aAPC33, we will be able to compare the clinical efficacy of T cells cultured for the short or long term.

Additional factors may influence the capacity of our CTL to expand and persist in vivo. It is possible that endogenous cytokine levels in the local microenvironment would support the persistence of CTL after reinfusion because our ex vivo cultures employ only low concentrations of IL-2 and IL-15. Alternatively, if additional signals are required, modest systemic doses of cytokine could be given, minimizing the possibility of severe side effects. Moreover, as has been shown in murine models, antigen-specific vaccination could be used to enhance the expansion and persistence of transferred CTL in vivo (47, 48).

aAPC unites many of the advantages shown by previously published artificial APC. Like artificial APC based on polystyrene beads or xenogeneic cells, aAPC33 delivers strong antigen-specific stimulation while minimizing negative regulatory signals. It is also an off-the-shelf, renewable source of APC that can be used to repeatedly stimulate antigen-specific CD8+ T cells ex vivo without exposure to xenogeneic antigens or to allogeneic HLA molecules. Importantly, we have avoided approaches requiring tetramer-guided cell sorting or priming with autologous DC because of feasibility and cost. The long-lived antigen-specific T cells generated possess a favorable phenotypic and functional profile that will be studied in clinical trials of adoptive transfer. Whether T cells generated by aAPC33 are superior to the T cells generated in other systems is unknown and warrants further examination. Although other clinical investigators have not found bystander T cells to induce significant toxicity (16, 49), we must assess the impact of these nonspecific T cells generated with our system in a clinical trial. We will also evaluate whether the clinical efficacy of our aAPC33-generated CTL can be enhanced by additional immune interventions such as preinfusion lymphodepletion, postinfusion vaccination, and cytokine administration, as well as other immunotherapeutic approaches.


   Acknowledgments
 
We thank John Daley, Suzan Lazo-Kallanian, and Mirra Chung for excellent technical help.


   Footnotes
 
Grant support: Clinical translation was partly supported by a grant from the Center for Human Cell Therapy. Also, NIH grant CA87720 (M.O. Butler), Dr. Mildred Scheel Stiftung der Deutschen Krebshilfe (S. Ansén), NIH grant CA92625-04, Cancer Research Institute grant (L.M. Nadler), and NIH grant HL54785-08 (N. Hirano).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

7 J.S. Lee and R.C. Mulligan, unpublished data. Back

Received 8/ 1/06; revised 12/ 5/06; accepted 1/ 2/07.


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 Materials and Methods
 Results
 Discussion
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