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T lymphocytes expressing a chimeric antigen receptor (CAR) targeting the CD19 antigen (CAR.19) may be of value for the therapy of B-cell malignancies. Because the in vivo survival, expansion and anti-lymphoma activity of CAR.19+ T cells remain suboptimal even when the CAR contains a CD28 costimulatory endodomain, we generated a novel construct that also incorporates the interleukin-15 (IL15) gene and an inducible caspase-9-based suicide gene (iC9/CAR.19/IL15). We found that compared to CAR.19+ T cells, iC9/CAR.19/IL15+ T cells had: (i) greater numeric expansion upon antigen stimulation (10-fold greater expansion in vitro, and 3 to 15 fold greater expansion in vivo) and reduced cell death rate (Annexin-V+/7-AAD+ cells 10% ± 6% for iC9/CAR.19/IL15+ T cells and 32% ± 19% CAR.19+ T cells); (ii) reduced expression of the programmed death 1 (PD-1) receptor upon antigen stimulation (PD-1+ cells <15% for iC9/CAR.19/IL15+ T cells versus >40% for CAR.19+ T cells); (iii) improved anti-tumor effects in vivo (from 4.7 to 5.4-fold reduced tumor growth). In addition, iC9/CAR.19/IL15+ T cells were efficiently eliminated upon pharmacologic activation of the suicide gene. In summary, this strategy safely increases the anti-lymphoma/leukemia effects of CAR.19-redirected T lymphocytes and may be a useful approach for treatment of patients with B-cell malignancies.
T lymphocytes expressing a chimeric antigen receptor (CAR) can be adoptively transferred to target a range of human malignancies including non-Hodgkin and Hodgkin lymphomas(1–5). CARs most commonly combine the antigen-binding specificity of a monoclonal antibody with the effector endodomain of the CD3/T-cell receptor complex (ζ chain), and redirect the specificity of T lymphocytes toward surface antigens expressed by tumor cells(6). CARs that target B-lineage restricted antigens such as CD19(7, 8), CD20(9), and the light chain of human immunoglubulins(10), or CD30 expressed by Reed-Sternberg cells(2, 4) have been cloned and validated in preclinical lymphoma/leukemia models, and some are currently in Phase I clinical trials(1, 3, 5, 11). However, it is evident from both clinical trials(1, 12, 13) and preclinical models(3, 10, 14) that the expansion and persistence of CAR-modified T cells in vivo are hampered by the lack of costimulatory signals following engagement with target antigens, since many tumor cells down regulate their expression of the costimulatory molecules required for optimal and sustained T-cell function, proliferation and persistence(3, 5).
This limitation has been partially resolved by the construction of “second generation” CARs in which a costimulatory endodomain derived from molecules such as CD28(10, 14, 15) or 4-1BB(16, 17) have been incorporated within the chimeric receptors. T cells expressing these enhanced CARs retain their cytotoxic function, but, upon antigen engagement, they produce IL2 which helps sustain their activation and expansion(10, 14, 15), and augments anti-tumor activity(3, 10, 14). To further potentiate the costimulation of CAR-modified T cells, “third generation” CARs have been developed which contain multiple costimulatory endodomains such as combinations of CD28 and 4-1BB(18–21) or CD28 and OX40(22), which may have superior activity compared to those encoding single costimulatory endodomains(18–20, 22).
We now describe an alternative strategy. We have engineered CAR-modified T cells to receive not only costimulation through the CD28 pathway but also to ectopically produce IL15, a cytokine crucial for T-cell homeostasis and survival(23, 24). Since these changes may increase the risk of direct toxicity and uncontrolled proliferation(25), we have also included a suicide gene that can be pharmacologically activated to eliminate transgenic cells as required(26, 27).
The following cell lines were used: Daudi and Raji (CD19+ Burkitt lymphoma cell lines), HDLM-2 (CD30+CD19− Hodgkin lymphoma cell line), Karpas-299 (CD30+CD19− anaplastic lymphoma cell line) and K562 (chronic erythroid leukemia cell line). All cells were purchased from ATCC and maintained in culture in RPMI 1640 (Gibco-BRL, San Francisco, CA) supplemented with 10% FBS (Hyclone, Waltham, MA) and and 2 mM L-glutamine (Gibco-BRL).
The cassette encoding the single chain antibody targeting CD19(28), the CD28 endodomain(10) and the ζ chain of the T-cell receptor complex(10) was cloned into the SFG retroviral backbone to generate the CAR.19 retroviral vector (Supplemental Fig. 1A). We then generated a second retroviral vector encoding the same CD19-specific CAR in combination with the human IL15 gene(27) and the inducible caspase-9 suicide gene that induces apoptosis upon specific binding with the small molecule dimerizer CID AP20187(26). The three genes were linked together using 2A sequence peptides derived from foot-and-mouth disease virus(27), and cloned into the SFG retroviral vector to generate the iC9/CAR.19/IL15 retroviral vector (Supplemental Fig. 1A). The vectors encoding FireFly Luciferase (FFLuc) and the fusion protein eGFP-FireFly luciferase (eGFP-FFLuc) used for in vivo imaging have been described previously(4, 10). Transient retroviral supernatants was produced as previously described(10).
Peripheral blood mononuclear cells (PBMC) were obtained from four healthy donors and three patients with chronic lymphocytic leukemia (B-CLL) as per local IRB approved protocols. PBMC or CD3+ enriched T cells (Miltenyi, Bergisch Gladbach, Germany) for samples collected from B-CLL patients(10) were activated with OKT3 (Ortho Biotech, Bridgewater, NJ) and CD28 (Becton Dickinson, Mountain View, CA) antibodies and recombinant human interleukin-2 (IL2) (100 U/mL) (Proleukin; Chiron, Emeryville, CA) in complete media [RPMI 1640 (Gibco-BRL) 45%, Click medium (Irvine Scientific, Santa Ana, CA) 45%, supplemented with 10% FCS (Hyclone) and 2 mM L-glutamine (GIBCO-BRL)](10). Activated T cells were transduced with retroviral supernatants on day 3 in plates coated with recombinant fibronectin fragment (FN CH-296; Retronectin; Takara Shuzo, Otsu, Japan)(10). After transduction, T cells were expanded using IL2 and then used for the experiments described below.
Seven days after transduction, control non-transduced (NT), CAR.19+ and iC9/CAR.19/IL15+ T cells (1 × 106 cells per well) were cocultured in 24 well plates with B-CLL cells (enriched-CD19+ cells) at an effector:tumor cell ratio (E:T) of 1:1. Culture supernatants were collected after 24, 48 and 72 hrs of culture to measure the production of IL2, IL15 and IFNγ using specific Elisas (R&D Systems, Inc, Minneapolis, MN).
To evaluate the T-cell growth, control NT, CAR.19+ and iC9/CAR.19/IL15+ T cells were maintained in culture and stimulated once a week with CD19+ B-CLL cells (E:T ratio 1:2) without any addition of exogenous cytokines. Cells were cultured for five weeks, and counted by trypan blue exclusion every week.
To measure the cell division of T cells upon antigen stimulation, we labeled control NT, CAR.19+ and iC9/CAR.19/IL15+ T cells with carboxyfluorescein diacetate succinimidyl ester (CFSE) (eBioscience, Inc. San Diego, CA)(29). We then stimulated the T cells with CD19+ B-CLL cells (E:T ratio of 2:1) and measured the CFSE dilution by FACS analysis after 5 days of culture. To measure T-cell death upon antigen stimulation we used the Annexin-V/7-AAD staining and FACS analysis(27).
To evaluate elimination of tumor cells, control NT, CAR.19+ and iC9/CAR.19/IL15+ T cells were cocultured with Daudi cells (CD19+) (E:T ratio of 5:1). After 3 days culture, cells were collected and residual tumor cells enumerated by FACS analysis. For samples obtained from B-CLL patients, anti-tumor effects were evaluated against autologous B-CLL cells (E:T ratio of 2:1). In these experiments, B-CLL cells were labeled with CFSE and enumerated by FACS after 3–4 days of coculture(10). In some experiments, we used wild type Karpas cells (CD30+CD19−) or CD19+ transgenic Karpas as the targets.
CID AP20187 (ARIAD Pharmaceuticals, Cambridge, MA) was kindly provided by Dr. Spencer (Baylor College of Medicine) and added at the indicated concentrations to T-cell cultures. The elimination of transgenic cells coexpressing the inducible suicide gene was evaluated 24–48 hours after incubation using Annexin-V/7-AAD staining and FACS analysis(27).
Cells were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or Peridinin-chlorophyll proteins (PerCP)-conjugated monoclonal antobodies (mAbs). To stain the tumor cells we used CD19, CD20 and CD30 from Becton Dickinson (Mountain View, CA). For the T lymphocytes we used CD3, CD4, CD8, CD56, CD45RA, CD45RO, CD62L, CD27, CD28, CCR7, Bcl-2 and PD-1 from Becton Dickinson (Mountain View, CA). To detect the expression of CAR.19, we used a mAb Fc-specific cyanine-Cy5-conjugated provided by Jackson ImmunoResearch (West Grove, PA), which recognized the IgG1-CH2CH3 component of the artificial receptor(10). Apoptosis was measured using Annexin-V and 7AAD staining (Becton Dickinson). Cells were analyzed by FACScan (Becton Dickinson), equipped with the filter set for triple fluorescence signals.
We used 4 hour 51Cr-release assays to evaluate the cytotoxic activity of control and CAR+ T lymphocytes(10). The labeled targets tested included Daudi (CD19+ target), HDLM-2 (CD19− target) and K562 (natural killer cell target).
To assess the persistence and anti-tumor effect of CAR+ T cells in vivo, we used a SCID-lymphoma human xenograft model. Mouse experiments were performed in accordance with Baylor College of Medicine Animal Husbandry guidelines according to an IACUC approved protocol.
In the first set of experiments to evaluate engraftment, Daudi and Raji cells were labeled with FFLuc. SCID mice (8–10 week old; Harlan Sprague Dawley Inc. Indianapolis, IN) were sublethally irradiated (250 rad) and injected intravenously (i.v) with either Daudi (3 ×106) or Raji (2 × 105). Tumor engraftment was measured using the in vivo imaging system as previously described(4, 10, 27). Briefly, mice were injected intraperitoneally (i.p.) with D-luciferin (150 mg/kg), and analyzed using the Xenogen-IVIS Imaging System. Signal intensity was measured as total photon/sec/cm2/sr (p/s/cm2/sr) as previously described(27, 29, 30) In the second set of experiments to evaluate the in vivo trafficking and persistence of CAR+ cells, either control or CAR.19+ or iC9/CAR.19/IL15+ cells were labeled with eGFP-FFLuc gene(4, 27). Seven days post engraftment with unlabeled Daudi or Raji cells, mice received i.v. 10 × 106 T cells. No exogenous cytokines were administered to the mice. Trafficking, persistence and expansion of labeled T cells were measured using the Xenogen-IVIS Imaging System(4, 10, 27).
To measure the anti-tumor effects of CAR+ T cells, mice were engrafted either i.p. or subcutaneously (s.c.) with Daudi cells (1 × 106 cells) labeled with the FFLuc gene. Ten days later, when the tumor was consistently measurable by light emission, mice received either control NT or CAR.19+ or iC9/CAR.19/IL15+ T cells (10 × 106; 2 doses, 1 week apart). For these experiments we used unlabeled T cells. We evaluated tumor growth using the Xenogen-IVIS Imaging System(4, 10, 27).
To evaluate the functionality of the suicide gene, mice bearing tumor cells and receiving iC9/CAR.19/IL15+ T cells labeled with the eGFP-FFluc gene, were treated with CID (50 µg) i.p. 2–3 doses every other day(27). CID treatment was initiated when the T-cell bioluminescent signal was exponentially increasing, indicating active expansion of the transgenic cells. Mice were then imaged as described above.
Student’s t test was used to determine the statistical significance of differences between samples, and P < .05 was accepted as indicating a significant difference. For the bioluminescence experiments, intensity signals were summarized using mean ± 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-ranks test(27, 29).
Activated T lymphocytes were equally transduced with one of two retroviral vectors encoding either CAR targeting the CD19 antigen and incorporating the CD28 endodomain (CAR.19) or the same CAR coexpressed with IL15 and the inducible suicide gene caspase-9 (iC9/CAR.19/IL15) (Supplemental Fig. 1B). We then measured IL15 production by iC9/CAR.19/IL15+ T cells and determined whether production was antigen dependent. Control NT, CAR.19+ and iC9/CAR.19/IL15+ T lymphocytes were cultured with or without CD19+ target cells (B-CLL)(10). Culture supernatants were collected at multiple time points to measure IL15 release. As shown in Fig. 1A, IL15 was undetectable in supernatants collected from stimulated or unstimulated control NT and CAR.19+ T cells. By contrast, iC9/CAR.19/IL15+ T cells produced small amounts of IL15 in the absence of antigen stimulation [25 pg/mL/106 cells (range 3 – 47 pg/mL)], which significantly increased 72 hours after antigen stimulation [240 pg/mL/106 cells (range 110 – 380 pg/mL)] (p<0.001). Importantly, when iC9/CAR.19/IL15+ T cells were maintained in culture for more than 4 weeks by weekly stimulation with CD19+ B-CLL cells, we found that the production of IL15 was sustained upon each antigen stimulation (Fig. 1B). Since CAR.19 contains the costimulatory endodomain CD28, we also measured IL2 production by genetically modified T cells(10, 14, 15). As shown in Fig. 1C, incorporation of IL15 within the iC9/CAR.19/IL15 vector did not compromise the production of IL2 by iC9/CAR.19/IL15+ T cells following antigen stimulation (mean 1527 pg/mL/106 cells, range 740 to 2000 for CAR.CD19+ cells and 1643 pg/mL/106 cells, range 631 to 2000 for iC9/CAR.19/IL15+ T cells) (p=0.8).
To evaluate whether IL15 and IL2 production by iC9/CAR.19/IL15+ T cells increased their expansion compared to CAR.19+ cells (which produce only IL2 in response to antigen stimulation) (Fig. 1A, C), we maintained both CAR.19+ and iC9/CAR.19/IL15+ T cells in culture by stimulating them weekly with CD19+ B-CLL cells. As shown in Fig. 1D, iC9/CAR.19/IL15+ T cell numbers increased 10 fold compared to CAR.19+ T cells (157 × 106 ± 66 ×106 total cells vs 15 × 106 ± 16 × 106 total cells, respectively) (p=0.005) after 5 weeks of culture. By contrast, neither CAR.CD19+ T cells nor iC9/CAR.19/IL15+ T cells significantly expanded in the absence of antigen stimulation (Fig. 1E). The viability of iC9/CAR.19/IL15+ T cells in the absence of antigen stimulation was, however, preserved long-term (4–5 weeks) compared to control NT or CAR.19+ T cells (Fig. 1E). Expanded CAR.19+ and iC9/CAR.19/IL15+ T cells contained both naïve (CD45RA+) and memory (CD45RO+) CD4+ and CD8+ T lymphocytes, with circa 20% of the latter retaining CD62L and CCR7 expression (data not shown).
To distinguish whether the greater number of iC9/CAR.19/IL15+ T cells compared to CAR.19+ T cells following antigen stimulation was due to increased proliferation or reduced cell death, we analyzed the proliferation and apoptosis of T cells upon antigen stimulation, using CFSE and Annexin-V/7-AAD based assays, respectively. To measure cell division, T cells were labeled with CFSE and then stimulated with CD19+ B-CLL cells. As shown in Fig. 2A, after 5 days of culture CFSE dilution was comparable for CAR.CD19+ and iC9/CAR.19/IL15+ T cells (77% ± 20% and 65% ± 20%, respectively, p=0.07), suggesting similar rates of cell division. By contrast, the death rate of iC9/CAR.19/IL15+ T cells was reduced 5 days after antigenic stimulation, as assessed by Annexin-V/7-AAD staining (annexin-V+/7-AAD+ cells were 32% ± 19% and 10% ± 6% for CAR.19+ and iC9/CAR.19/IL15+ T cells, respectively) (p<0.001) (Fig. 2B). The improved viability of transgenic cells producing IL15 correlated also with an increased expression of anti-apoptotic genes, such as Bcl-2 (Fig. 2C).
To evaluate the trafficking and persistence of our modified T cells in vivo, we used a SCID mouse lymphoma xenograft, and an extensively validated bioluminescence imaging system(4, 27, 29). We began by evaluating the tumor engraftment after i.v. inoculation either of Daudi and Raji, both CD19+ lymphoma cell lines, labeled with FFLuc. We found that 5 days after infusion, Daudi (3 × 106 cells) had engrafted diffusely in bone marrow, lymphnodes and spleen (Fig. 3A), while Raji (2 × 105 cells) had preferentially engrafted in the spinal cord (Fig. 3D). Tumor localization was confirmed by phenotypic analysis of biopsy samples (data not shown). After defining the timing and sites of tumor engraftment, we assessed T-cell trafficking to the tumor and T-cell persistence in vivo. Control NT, CAR.19+ and iC9/CAR.19/IL15+ T cells were labeled with eGFP-FFLuc(10, 27), and infused (10 × 106 cells/mouse) in mice previously engrafted with either unlabelled Daudi or Raji cells. Figs. 3B and E illustrate that both CAR.19+ and iC9/CAR.19/IL15+ T cells localized at the tumor site since T-cell bioluminescence had superimposable anatomical localizations for the labeled tumor cells (Figs. 3A and D). T-cell bioluminescence remained barely detectable when Daudi- or Raji-engrafted tumor cells were treated with labeled control NT T cells (data not shown). Importantly, while the bioluminescence signal corresponding to CAR.19+ T cells only modestly increased by day 25 post infusion (from 4 × 105 ± 8 × 103 to 4 × 105 ± 4 × 104 for mice engrafted with Daudi, and from 1 × 105 ± 2 × 104 to 1.2 × 105 ± 3 × 104 for mice engrafted with Raji) corresponding to 1.1 and 1 fold increase, respectively (Figs. 3C and F), the signal from iC9/CAR.19/IL15+ T cells significantly increased over the following 25 days (from 5 × 105 ± 8 × 104 to 9 × 106 ± 3 × 106 for mice engrafted with Daudi and from 5 × 105 ± 8 × 104 to 2 × 106 ± 1 × 106 for mice engrafted with Raji), corresponding to 15 and 3 fold increase, respectively (p=0.02 and p=0.01) (Figs. 3C and F), showing increased expansion and persistence of these cells.
We measured the cytotoxic activity of T cells against CD19+ and CD19− tumor cell lines using standard 51Cr release assays. Both CAR.CD19+ and iC9/CAR.19/IL15+ T cells had equal and specific cytotoxic activity against CD19+ Daudi cells (63% ± 17% and 57% ± 16% specific lysis at an E:T ratio of 20:1, respectively), with less than 13% killing of HDLM-2 (CD19−) and erythroleukemia derived K562 (NK cell target) cell lines (Fig. 4A). Control T cells showed no significant cytotoxic activity against any of these target cell lines. In parallel, both CAR.19+ and iC9/CAR.19/IL15+ T cells produced equal amounts of INFγ in response to CD19+ tumor cells (mean of 8327 pg/mL/106 cells, range 5080 to 12510 for CAR.CD19+ cells and of 9147 pg/mL/106 cells, range 3025 to 18010 for iC9/CAR.19/IL15+ cells) (Fig. 4B). To further confirm that IL15 production by iC9/CAR.19/IL15+ T cells enhanced the elimination of tumor cells through the CAR, we cocultured T cells with wild type Karpas-299 tumor cells (CD30+CD19−), or with Karpas cells modified to stably express the CD19 molecule (Karpas CD30+CD19+). After 4–5 days at an initial T cell:tumor cell ratio of 5:1, residual tumor cells were quantified by FACS analysis enumerating CD30+ tumor cells. Both CAR.19+ and iC9/CAR.19/IL15+ T cells efficiently eliminated Karpas CD19+ tumor cells when T-cell anti-tumor activity was evaluated using T-cell lines maintained in short term culture (1 week) following transduction (data not shown). By contrast, when T-cell lines were maintained in culture for 4 weeks, and stimulated weekly with CD19+ B-CLL cells, only iC9/CAR.19/IL15+ T cells maintained their ability to completely eliminate tumor cells from the culture (residual tumor cells 1% ± 0.7% and 10% ± 5% for iC9/CAR.19/IL15+ T cells and CAR.19+ T cells, respectively) (p=0.001). Importantly, even after 4 weeks expansion in culture, the anti-tumor effects of both CAR.19+ and iC9/CAR.19/IL15+ T cells remained antigen-specific, since they lacked activity against wild type (CD19−) Karpas-299 cells (Fig. 4C).
To discover potential mechanisms for the sustained effector function of IL15-producing cells upon prolonged culture and repeated antigen stimulation, we evaluated the expression of PD-1, a marker of T-cell exhaustion(31), and found that iC9/CAR.19/IL15+ T cells had lower expression of PD-1 (PD-1+ T cells <15%) two days after stimulation with B-CLL cells than CAR.19+ T cells (PD-1+ T cells >40%) (Fig. 4D).
The above in vitro data were then corroborated with experiments in vivo. In two different models, SCID mice were engrafted either i.p. or s.c. with 3 × 106 Daudi cells labeled with FFLuc. Seven days later, these mice were treated with 2 weekly infusions i.p. (for mice engrafted with i.p. tumor.) or i.v (for mice engrafted with s.c. tumor) of control NT, CAR.19+ or iC9/CAR.19/IL15+ T cells (10 × 106). Tumor growth was monitored by measuring changes in tumor bioluminescence over time. As shown in Figs. 5A, B, the tumor bioluminescence of mice engrafted i.p. with Daudi cells rapidly increased in recipients of control NT T cells (rising from 1.8 × 108 ± 4 × 107 to 16 × 108 ± 2.6 × 108 by day 38). CAR.19+ T cells transiently controlled tumor growth (from 1.9 × 108 ± 3 × 107 to 9.3 × 108 ± 1.6 × 108 by day 38), while iC9/CAR.19/IL15+ T cells significantly controlled tumor expansion so that signal rose from 1.6 × 108 ± 3 × 107 to only 1.7 × 108 ± 5 × 108 by day 38 (p=0.001 when compared with mice receiving CAR.19+ T cells). Similarly, the tumor bioluminescence of mice engrafted s.c. with Daudi cells rapidly increased in mice receiving control NT T cells (from 2.6 × 105 ± 1.1 × 104 to 57 × 107 ± 20 × 107 by day 24), showed transient control in recipients of CAR.19+ T cells (from 3.3 × 105 ± 9.6 × 104 to 26 × 107 ± 7.6 × 107 by day 24) and greatest control in recipients of iC9/CAR.19/IL15+ T cells (tumor growth from 2.8 × 105 ± 7 × 104 to 5.5 × 107 ± 1.5 × 107 by day 24) (p=0.02 when compared with mice receiving CAR.19+ T cells) (Figs. 5C, D).
Because the production of an autocrine growth factor raises concerns about autonomous, uncontrolled T-cell growth, we incorporated in our construct a suicide gene based on the inducible caspase-9 gene(26, 27). As shown in Fig. 6A, the addition of 50 nM CID to cultures of iC9/CAR.19/IL15+ T cells induced apoptosis/necrosis of more than >95% of transgenic cells within 24 hours as assessed by annexin-V-7AAD staining(27). The suicide gene was also effective in vivo. Mice were engrafted i.v. with Raji tumor cells and then infused with eGFP-FFLuc labeled iC9/CAR.19/IL15+ T cells. These cells localized and expanded at the tumor site by day 14 after infusion as assessed by bioluminescence measurement (Fig. 6B). T-cell bioluminescence drastically reduced (from 1.2 ×106 ± 7.7 × 105 to 1.3 × 105 ± 3 × 104) following administration of the CID, consistent with a significant elimination of the transgenic cells(27).
Finally, we validated the efficacy of the combination of CAR.19, IL15 and the suicide gene in cells obtained from subjects with B-CLL. As shown in Fig. 7A, the expansion of T cells isolated from B-CLL patients was enhanced following transduction with the iC9/CAR.19/IL15 vector and stimulation with autologous B-CLL cells (E:T ratio 1:1) compared to CAR.19+ T cells (228 × 106 ± 315 × 106 total cells vs 7 × 106 ± 2.7 × 106 total cells, respectively). Their expansion remained fully antigen dependent as we observed for T-cell lines generated from healthy donors (Fig. 7B). iC9/CAR.19/IL15+ T cells also released both IL15 (103 ± 39 pg/mL/106 cells) and IL2 (62 ± 24 pg/mL/106 cells) in response to autologous B-CLL cells, while CAR.19+ T cells produced only IL2 (65 ± 33 pg/mL/106 cells) (Fig. 7C). Expansion of patients’ T cells was mainly driven by the antiapoptotic effect of IL15 upon antigen stimulation (Fig. 7D). We also confirmed that iC9/CAR.19/IL15+ patient T cells retained enhanced anti-tumor activity against autologous B-CLL after long-term culture and repeated exposure to autologous tumor cells, while CAR.19+ patient T cells did not (Fig. 7E).
We have forced expression of IL15 in T cells redirected with a CAR that specifically targets the CD19 antigen, and demonstrated that these engineered T cells had superior survival, expansion and anti-tumor activity in vivo compared to redirected T cells that only receive CD28 costimulation through the CAR but lack IL15 production. Importantly, the incorporation of an inducible suicide gene and its pharmacologic activation efficiently eliminated these gene modified T cells further increasing the safety of the proposed approach.
In vivo persistence and expansion of adoptively transferred tumor-specific T cells is crucial to obtain sustained clinical responses(32). This is particularly important for T cells engrafted with CARs that lack costimulatory endodomains since they do not release helper cytokines upon engagement with the antigen expressed by tumor cells(10, 14, 15, 33, 34). In addition, they cannot receive appropriate activation by professional antigen presenting cells (APCs) in secondary lymphoid organs, since the native αβT-cell receptors of these redirected T cells are not generally specific for latent antigens consistently processed and presented by the host APCs(4, 13). The incorporation of the CD28 costimulatory signaling domain as part of the CAR itself(10, 14, 15, 33) can enhance activation and proliferation of these cells secondary to IL2 secretion, even without cross-presentation by APCs(10, 15). However, the overall persistence and anti-tumor effects of such CAR.19-redirectd T cells still remain limited(10, 14, 19). Alternative costimulatory endodomains may be superior to CD28, and incorporation of 4-1BB endodomain for example(16, 17) or a combination of both CD28 and 4-1BB has been reported to increase the persistence and anti-tumor efficacy over CD28 containing CARs(18–21). Nevertheless, we reasoned that the ectopic production of IL15 by CAR-modified T cells may represent a valid addition to the incorporation of costimulatory endodomains within the CAR construct since the benefits would occur through different pathways and mechanisms, since neither CD28 nor 4-1BB costimulation lead to the production of IL15, a cytokine that potently enhances the anti-tumor activity of effector T cells in vitro and in vivo(8, 35, 36). Our approach ensures that CAR-modified T cells receive both IL2 and IL15 stimulation following chimeric receptor engagement in the tumor microenvironment.
We have accommodated the IL15 gene in a single retrovirus vector in combination with the CAR.19 encoding the CD28 endodomain and an inducible suicide gene without significantly affecting CAR.19 expression, an essential requirement if tumor-specificity is to be maintained. Minimal cytokine is produced when the T cells are unstimulated, but the amount significantly increases after stimulation with tumor cells, and production is maintained by the T cells after more than 4 weeks culture. This transgenic IL15 is biologically functional, as iC9/CAR.19/IL15+ T cells have superior expansion following antigen stimulation than control CAR.19+ T cells. These benefits are largely attributable to the reduced susceptibility of iC9/CAR.19/IL15+ T cells to cell death induced upon antigen stimulation, likely because these cells have higher expression of the antiapoptotic gene Bcl-2(37) as compared to control CAR.19+ T cells. Importantly, our in vitro experiments demonstrate that iC9/CAR.19/IL15+ T cells retain enhanced anti-tumor activity when they are “chronically” exposed to tumor cells. This effect may be determined by an increased protection of iC9/CAR.19/IL15+ T cells from functional exhaustion, as indicated by a lower expression of PD-1 upon antigen-stimulation than CAR.19+ T cells, since PD-1 has been recognized as a marker of exhausted T cells in chronic infections such as HIV(31) and hepatitis C(38), and tumor infiltrating T lymphocytes(39–42). We do not yet know the transcriptional or post-transcriptional mechanisms that reduce expression of PD-1 in iC9/CAR.19/IL15+ T cells after repeated antigen stimulation. Nonetheless, our observation may have clinical implications since CAR-modified T cells that target self-antigens such as CD19, will inevitably be exposed to a large number of target cells in vivo, and PD-1 ligands may be expressed either by tumor cells itself(39, 41, 42) or by tumor-associated dendritic cells(43). The improved anti-tumor effects observed in vitro were matched by superior in vivo activity, and iC9/CAR.19/IL15+ T cells retained their tumor-homing, expanded at tumor sites and had enhanced anti-tumor activity compared to control CAR.19+ T cells.
Recently, intermittent systemic administration of recombinant IL15 has been demonstrated to induce expansion of memory CD4+ and CD8+ T cells in nonhuman primates(44). Administration of IL15 will soon be tested in patients to support the expansion of adoptively transferred tumor-specific T cells. We suggest that our proposed approach may remain advantageous as it delivers the cytokine directly to T cells at the tumor site, avoiding the toxicities that may be observed in patients receiving systemic administration of recombinant cytokines, especially when the cytokine is used at high doses(45). Our approach will also remain valuable for adoptive transfer of tumor-specific T lymphocytes in lymphodepleted patients(46) as it ensures long term availability of IL15 for tumor-specific T cells to overcome the antiproliferative effect of TGF-β(47) and regulatory T cells(48) (and manuscript in preparation) within the tumor microenvironment.
Because IL15 production by gene modified T cells occurs predominantly after they engage tumor cells through their CAR, the risks of autonomous growth should remain small. Nonetheless, given concerns with retroviral-associated oncogenesis(25), and the potential side effects reported after T-cell therapy with CAR-modified T cells(11, 49), we incorporated a suicide gene based on the inducible caspase-9 molecule within our construct. We demonstrated that the activation of this suicide gene rapidly induces apoptosis of IL15 producing T cells both in vitro and in vivo. Our previous observation that the small fraction of cells (<10%) that seems to escape apoptosis/necrosis shortly after exposure to the dimerizer drug do not express detectable levels of any transgene including cytokine(27), nor proliferate following antigen stimulation(27), may further increase the safety of the proposed approach. This suicide gene is already under evaluation in a Phase I clinical trial in which patients undergoing haploidentical transplant receive donor derived T cells gene modified with the inducible caspase-9 gene(50).
In conclusion, we show that transgenic expression of IL15 improves survival, expansion and anti-tumor effects of CD19-specific redirected T cells. The incorporation of an effective suicide gene should further assure the safety of the approach and increase its potential clinical applicability.
This work was supported in part from Leukemia and Lymphoma Society Specialized Center of Research (SCOR; grant no. 7018), NIH PO1CA94237, NIH P50CA126752, NIH RO1CA131027, Leukemia and Lymphoma Society Translational Research grants, Doris Duke Charitable Foundation/Clinical Scientist development award and CLL Global Research Foundation.
AUTHORSHIP. V.H., B.S, C.Q., M.Z., and J.V. performed the experiments. A.M. and B.S. performed the animal experiments. G.D., B.S., and V.H. designed the research and analyzed the data. V.H., B.S., and G.D wrote the manuscript. H.E.H, C.M.R. and M.K.B. critically reviewed the manuscript. All authors approved the final version of the manuscript.
DISCLOSURE. The authors declare no competing financial interests.