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Blood, 15 October 2007, Vol. 110, No. 8, pp. 2793-2802. Prepublished online as a Blood First Edition Paper on July 17, 2007; DOI 10.1182/blood-2007-02-072843.
PLENARY PAPER Co-expression of cytokine and suicide genes to enhance the activity and safety of tumor-specific cytotoxic T lymphocytes1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX
The antitumor effect of adoptively transferred tumor-specific cytotoxic T lymphocytes (CTLs) is impaired by the limited capacity of these cells to expand within the tumor microenvironment. Administration of interleukin 2 (IL-2) has been used to overcome this limitation, but the systemic toxicity and the expansion of unwanted cells, including regulatory T cells, limit the clinical value of this strategy. To discover whether transgenic expression of lymphokines by the CTLs themselves might overcome these limitations, we evaluated the effects of transgenic expression of IL-2 and IL-15 in our model of Epstein Barr Virus–specific CTLs (EBV-CTLs). We found that transgenic expression of IL-2 or IL-15 increased the expansion of EBV-CTLs both in vitro and in vivo in a severe combined immunodeficiency disease (SCID) mouse model and enhanced antitumor activity. Although the proliferation of these cytokine genes transduced CTLs remained strictly antigen dependent, clinical application of this approach likely requires the inclusion of a suicide gene to deal with the potential development of T-cell mutants with autonomous growth. We found that the incorporation of an inducible caspase-9 suicide gene allowed efficient elimination of transgenic CTLs after exposure to a chemical inducer of dimerization, thereby increasing the safety and feasibility of the approach.
Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) has shown efficacy in some patients with melanoma,1,2 Hodgkin lymphoma,3,4 and nasopharyngeal carcinoma.5,6 However, the antitumor activity of adoptively transferred CTLs is hampered by the limited capacity of these cells to significantly expand within the tumor microenvironment.7 The development of strategies to overcome this restriction could significantly improve the clinical outcome of patients receiving adoptive T-cell therapy. Since adoptively transferred antigen-specific CTLs are highly dependent on exogenous cytokines for their continued growth and survival,8 systemic administration of interleukin 2 (IL-2) has been used to enhance their in vivo expansion and persistence.1 However, the prolonged administration of IL-2 is often associated with serious side effects, limiting the amount and duration of cytokine administration.1 Moreover, the effects of systemically administered cytokines are nonselective. IL-2 may favor the expansion of unwanted cell subsets, such as regulatory T cells,9 that constitutively express the IL-2 receptor and adversely affect the function of antitumor CTLs.10–12 Genetic manipulation of CTLs to express growth cytokines such as IL-2 and IL-15 could make them less helper-cell dependent and better able to sustain their proliferation and activation after antigenic stimulation.13,14 However, the constitutive expression of transgenes that enhance growth raises the concern that the T cells may lose antigen specificity and growth dependence and become growth autonomous.15 This is a particular concern where retroviruses are used to ensure transgene integration and thereby obtain cytokine secretion by the progeny of the modified cells.16,17 We therefore used our Epstein Barr virus (EBV)+ tumor model and EBV-specific CTLs (EBV-CTLs) to evaluate the biological effects of transgenic expression of IL-2 or IL-15, in association with transfer of a suicide gene based on an inducible caspase-9 (iCasp-9) protein18 that can be activated using a specific chemical inducer of dimerization (CID), analogs of which have been safely tested in a phase I study.19 We found that both transgenic IL-2 and IL-15 sustained CTL expansion and function in vivo and that these cytokine-gene modified cells retained antigen specificity and dependence. Activation of the iCasp-9 gene with CID efficiently ablated cytokine production and eliminated adoptively transferred T cells, suggesting that this combined approach could safely augment the efficacy of adoptively transferred tumor-specific CTLs.
Human and animal studies were approved by the Institutional Review Board of Baylor College of Medicine. Plasmid construction and retrovirus production
CD34 was used as a selectable marker of transduced cells. Full-length human CD34 (NCBI AF523361) was cloned by polymerase chain reaction (PCR) using clone ID 4746591 (Invitrogen, Carlsbad, CA). We truncated the cytoplasmic tail of CD34, 20 amino acids (YYT) downstream from the last amino acid of the putative transmembrane domain. This truncation was based on preliminary experiments showing stable expression of
Generation and transduction of EBV-CTLs EBV-CTLs were prepared by stimulating peripheral blood mononuclear cells (PBMCs) with gamma-irradiated (40 Gy) autologous EBV-transformed lymphoblastoid cell lines (LCLs) on day 0 and + 9, then weekly thereafter. Recombinant human interleukin-2 (rhIL-2) (50 U/mL) (Proleukin; Chiron, Emeryville, CA) was added twice a week from day 14 as previously described.27 For transduction, EBV-CTLs obtained after at least 3 stimulations were plated at 0.5 x 106 cells/well in 24-well plates precoated with recombinant fibronectin fragment (FN CH-296; Retronectin; Takara Shuzo, Otsu, Japan) and incubated with the retroviral supernatant.25 Three days after transduction, EBV-CTLs were collected and then stimulated weekly with autologous LCLs, with or without the addition of exogenous cytokines rhIL-2 (50 U/mL) or recombinant human IL-15 (rhIL-15) (10 ng/mL) (R&D Systems, Minneapolis, MN). The release of IL-2 and IL-15 by control and transgenic CTLs was measured in the culture supernatant by specific ELISAs from R&D Systems. Immunophenotyping Cells were stained with phycoerythrin (PE)-, fluorescein isothiocyanate (FITC)–, or PerCP-conjugated monoclonal antibodies (MAbs). We used CD3, CD4, CD8, CD56, CD34, CD45RA, CD45RO, and CD62L from Becton Dickinson (Mountain View, CA) and MAbs specific for the TCR-Vß repertoire (IOTest ßMark kit; Immunotech, Emeryville, CA). Tetramers targeting known major histocompatibility complex (MHC) class I epitopes of EBV-related antigens also were used.25,28 The induction of apoptosis of EBV-CTLs was evaluated using the Annexin-V/7-AAD staining (Becton Dickinson). In some experiments Annexin-V+ CTLs were selected using Annexin-V-FITC antibody and anti-FITC magnetic beads (Miltenyi). Cells were analyzed by a FACScan (Becton Dickinson) equipped with the filter set for triple fluorescence signals. Activation of the suicide gene
The chemical inducer of dimerization (CID) (AP20187; ARIAD Pharmaceuticals, Cambridge, MA) was kindly provided by Dr Spencer (Baylor College of Medicine) and added at the indicated concentrations to EBV-CTLs transduced either with Chromium release assay We evaluated the cytotoxic activity of EBV-CTLs by using a standard 4-hour 51Cr release assay, as previously described.29 As target cells we used autologous LCLs, HLA class I and II mismatched LCLs, as well as the HSB-2 and K562 cell lines that measure lymphokine-activated and natural killer activity, respectively. Target cells incubated in media alone or in 1% Triton X-100 were used to determine spontaneous and maximum 51Cr release, respectively. The mean percentage of specific lysis of triplicate wells was calculated as follows: [(test counts - spontaneous counts)/(maximum counts - spontaneous counts)] x 100. Enzyme-linked immunospot assay
The interferon Western blot analysis Cell lysates were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Caspase-9 and CD34 proteins were detected by immunoblot using specific monoclonal Abs (Abcam Inc, Cambridge, MA). Immunoblots were developed using enhanced chemiluminescence detection reagents (Amersham Biosciences, Freiburg, Germany). Membranes were reprobed using the monoclonal anti-GAPDH Ab (Santa Cruz Biotechnologies, Santa Cruz, CA). In vivo study using a xenogenic SCID mouse model To assess the expansion, persistence, and antitumor effect of transgenic EBV-CTLs in vivo, we used a SCID mouse model and an in vivo imaging system. CB17 SCID mice 8 to 10 weeks old were purchased from Harland Sprague Dawley, Indianapolis, IN. Mouse experiments were performed in accordance with Baylor College of Medicine Animal Husbandry guidelines. In vivo expansion and antitumor effects of transgenic CTLs
To evaluate the in vivo expansion of transgenic CTLs, EBV-CTLs transduced either with iC. In vivo validation of the iCasp-9 suicide gene
To evaluate the functionality of the suicide gene, mice bearing LCLs and receiving EBV-CTLs transduced either with iC. Statistical analysis All in vitro data are presented as mean plus or minus 1 SD. Student t test was used to determine the statistical significance of differences between samples, and P less than .05 was accepted as indicating a significant difference. For the bioluminescent experiments, intensity signals were log-transformed and summarized using mean plus or minus SD at baseline and multiple subsequent time points for each group of mice. Changes in intensity of signal from baseline at each time point were calculated and compared using the Wilcoxon signed-rank test. Tumor-free survival was analyzed by Kaplan-Meier analysis (using SPSS software, SPSS, Chicago, IL), and the statistical significance of observed differences was assessed by log-rank and Breslow testing.
Transgenic expression of IL-2 and IL-15 sustains the expansion of EBV-CTLs
Figure 1A shows the construction schema for the vectors SFG.iCasp-9.2A.
We also evaluated the kinetics of IL-2 and IL-15 cytokine production by CTLs transduced either with iC.
We next evaluated whether the quantity of cytokine production was sufficient to sustain the expansion of transgenic EBV-CTLs in response to specific antigenic stimulation. After transduction, EBV-CTLs were maintained in culture and stimulated once a week with autologous LCLs (E/T ratio of 1:1) without addition of exogenous cytokines. In parallel, control EBV-CTLs transduced with
IL-2 and IL-15 transgenic CTLs retain their antigen specificity
To confirm that the genetic manipulation of EBV-CTLs did not modify their antigen specificity, we monitored their phenotype, polyclonality, and antigen repertoire over 5 to 6 weeks of culture. The phenotypic profile of CTLs was not altered by cytokine transduction, as most EBV-CTLs remained CD3+/CD8+ (96% ± 1%, 97% ± 2%, and 98% ± 2% for
To further demonstrate that EBV-CTLs maintained the pattern of antigen response after transduction, we evaluated the frequency with which each EBV-CTL population recognized HLA class I–restricted EBV peptides, using specific peptide HLA tetramers and flow cytometric analysis to measure binding and IFN  ELIspots to measure responsiveness. As shown in Figure 3C,D and Table 1, the frequency of EBV-specific CTLs in control and transgenic CTLs was retained. Although the distribution of antigen specificity and Vß T-cell receptor ( ßTCR) drifted over time, there was continued polyclonality of the transgenic EBV-CTLs, with no evidence for progressive clonal outgrowth compared with control CTLs growing with exogenous rhIL-2 (Table S1).
Expression of transgenic cytokines improves in vivo expansion and antitumor activity of EBV-specific CTLs
To assess the in vivo functionality of transgenic CTLs, we used a SCID mouse tumor model, in which the animals were engrafted subcutaneously with EBV-LCLs and then infused intravenously with autologous EBV-CTLs.33 Transduced EBV-CTLs obtained from 4 healthy donors were selected with anti-CD34 microbeads and then transduced with the vector encoding eGFP-FFLuc.24 After the second transduction, the percentage of GFP+ EBV-CTLs ranged from 43% to 64% for
The increase of bioluminescence from IL-2 and IL-15 transgenic CTLs remained antigen dependent and MHC restricted in vivo: when the mice were engrafted with allogeneic EBV-LCLs, transgenic CTLs homed to the tumor but, unlike with autologous EBV-LCLs, did not expand significantly (Figure 4B). The increase in the bioluminescence signal for the cytokine-transgenic CTLs corresponded to an actual increase in number of CTLs accumulated within the tumor and not just to an increase in bioluminescence due to enhanced expression by the same number of cells. Figure 4C shows the increased CTL numbers in tumors excised from animals in the high (cytokine-transduced) versus the low (control) bioluminescence groups.
The increased expansion in vivo of cytokine transgenic CTLs was associated with an enhanced antitumor effect, as in both the IL-2 and the IL-15 transgenic CTL groups, 47% to 53% of the mice were tumor free 4 to 6 weeks after adoptive T-cell transfer, compared with none of the mice receiving control CTLs (P = .001 for iC.
Activation of the iCasp-9 suicide gene eliminates transgenic EBV-CTLs ex vivo and in vivo
For clinical applications, constitutive expression of transgenic cytokines would likely raise concerns about autonomous and uncontrolled growth of CTLs.16,17 We therefore incorporated the iCasp-9 suicide gene in our cytokine encoding retroviral vectors.18 This proapoptotic gene product is activated after exposure to a small CID (AP20187), which is an analog of FK506. Addition of CID (50 nM) to cultures of EBV-CTLs transduced either with iC.
The iCasp-9 system also was functional in vivo. iC.  CD34/IL-2v or iC.CD34/IL-15v–transduced EBV-CTLs were selected for CD34 expression and then transduced with the eGFP-FFLuc gene, as described above. Ten million EBV-CTLs were injected intravenously in tumor-bearing SCID mice. Once CTL expansion was detectable at the tumor site (assessed by progressively rising photon emission), the animals were injected intraperitoneally with CID. As illustrated in Figure 7A,B, photon emission at the time of CTL localization at the tumor site ranged from 5 x 104 to 105 p/s/cm2/sr, increasing to 1.8 x 106 to 1.5 x 107 p/s/cm2/sr (P < .01) by days 10 to 15. Mice were then treated with CID (50 µg) and the bioluminescence fell by more than 1 log (6.3 x 104 to 5.2 x 105 p/s/cm2/sr) within 24 to 72 hours, returning to the pre-expansion level (P = .1), compared with the bioluminescence at the time of CTL localization. In contrast, the bioluminescence increased over time in mice infused with IL-2 or IL-15 transgenic CTLs without the CID (Figure 7A,B). As shown in Figure 7C, the reduction of the bioluminescence after CID administration corresponded to a significant decrease in CTLs infiltrating the tumor, assessed by FACS analysis of biopsied tumors.
We have used retroviral mediated transgenic expression of IL-2 or IL-15 in tumor-specific CTLs to improve their expansion and efficacy. We also determined whether the inclusion of a suicide gene could remove the transgenic cells, to further increase safety should unwanted T-cell proliferation or autonomy occur. Using EBV as our model system, we found that IL-2 or IL-15 transgenic EBV-CTLs have enhanced expansion ex vivo and in vivo and retained their antigen specificity and effector function. Their improved survival and expansion were associated with increased antitumor activity in vivo. Importantly, we found that the growth of cytokine transgenic CTLs remained dependent on antigen stimulation and that the pharmacologic activation of a suicide gene iCasp-9 efficiently eliminated cytokine-producing CTLs. Preclinical and clinical trials of adoptive transfer with antitumor CTLs have shown that the limited in vivo expansion of transferred CTLs is one of the barriers that needs to be overcome to improve the clinical outcome of this therapeutic approach.7,34 Transgenic expression of growth-promoting cytokines by the adoptively transferred CTLs represents a strategy to support their own expansion/persistence.13,14 In addition, the localized production of cytokines by transgenic cells might avoid the toxicity of systemic administration of recombinant cytokines and reduce the expansion of other cells, such as regulatory T cells that impair the antitumor immune response.7,9,11,12 However, before translating this genetic modification in a clinical application, several issues need to be addressed, such as confirming that progeny of genetically modified cells maintain antigen specificity and dependence, providing evidence that genetically modified CTLs possess enhanced antitumor effects, and ensuring that these cells remain safe. Our model of EBV-tumor and EBV-CTLs demonstrates that retroviral gene transfer of IL-2 or IL-15 produces sufficient cytokines to sustain CTL expansion ex vivo and in vivo for more than 5 weeks, providing an improved antitumor effect. Importantly, the antigen specificity and dependence, and the MHC restriction of the CTL progeny, were maintained. Although extensive ex vivo T-cell culture can alter the antigenic and TCR repertoire of CTL lines, we found that IL-2 and IL-15 transgenic CTL lines retained broad reactivity to EBV epitopes and showed no progressive restriction of their antigen specificity or TCR repertoire compared with control CTL lines. In a previously described immunocompetent mouse model, the presence of IL-15 resulted in the polarization to central memory phenotype of adoptively transferred CTLs and improved antitumor effects compared with IL-2.35 In our model, we did not find differences in antitumor effects between IL-2 or IL-15 transgenic EBV-CTLs or a polarization to central memory phenotype in IL-15 transgenic CTLs. Since the gene transfer was performed in CTL lines obtained after 2 to 3 stimulations ex vivo, in cells exposed to IL-2 for 10 to 15 days, it is possible that at the time of transduction, cells are already polarized to an effector memory phenotype and that the transgenic production of IL-15 cannot then affect this phenotype.
The use of cytokine-expressing CTLs in clinical adoptive transfer raises potential concerns, since leukemic transformation or immortalization of T cells has been reported in a mouse model engrafted with murine T-cell lines expressing IL-2,36 in IL-15 transgenic mice,37 and in a human T-cell clone.14 By contrast, our study of cytokine-expressing EBV-CTLs failed to show any evidence for progressive clonality or for the development of antigen-independent proliferation. In the absence of antigen, the transgenic CTLs did not expand and died within 2 to 3 weeks. Moreover, we found that the bioluminescence signal from transgenic CTLs progressively declined in vivo after elimination of the tumor. This is likely related to the fact that transgenic cytokine production depends on the activation of the retroviral LTR, which in turn is strictly dependent on the activation status of the T cells and is optimal only in the presence of adequate
Although our observations support the safety of transgenic expression of cytokines in antigen-specific CTLs, we cannot exclude rare oncogenic events associated with retroviral integration near promoters or genes involved with critical elements of growth and survival.14,16,17 We therefore incorporated a suicide gene based on the inducible caspase-9 molecule within our constructs. To generate these retroviral vectors encoding for 3 genes, we coexpressed iCasp-9,
The inclusion of the selectable marker within the retroviral cassette might be useful for the enrichment of transgenic CTLs for a clinical trial. We used a truncated form of human CD34 since CD34+ selection can be performed using clinical grade reagents. However, In conclusion, the data we report specifically support the use of transgenic cytokines to improve the expansion and antitumor effects of antigen specific CTLs. The incorporation of an effective suicide gene should further increase the safety of the approach and increase its potential clinical applicability.
Contribution: C.Q. and J.V. contributed equally to the work; C.Q., J.V., B.S, G.G., and G.D. designed and performed the experiments; J.V., M.P., and G.D. designed and con | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
