Ex vivo Akt inhibition promotes the generation of potent CD19CAR T cells for adoptive immunotherapy
- Ryan Urak1,
- Miriam Walter1,
- Laura Lim1,
- ChingLam W. Wong1,
- Lihua E. Budde1,
- Sandra Thomas1,
- Stephen J. Forman†1 and
- Xiuli Wang†1Email author
© The Author(s). 2017
Received: 2 June 2016
Accepted: 17 February 2017
Published: 21 March 2017
Insufficient persistence and effector function of chimeric antigen receptor (CAR)-redirected T cells have been challenging issues for adoptive T cell therapy. Generating potent CAR T cells is of increasing importance in the field. Studies have demonstrated the importance of the Akt pathway in the regulation of T cell differentiation and memory formation. We now investigate whether inhibition of Akt signaling during ex vivo expansion of CAR T cells can promote the generation of CAR T cells with enhanced antitumor activity following adoptive therapy in a murine leukemia xenograft model.
Various T cell subsets including CD8+ T cells, bulk T cells, central memory T cells and naïve/memory T cells were isolated from PBMC of healthy donors, activated with CD3/CD28 beads, and transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain. The transduced CD19CAR T cells were expanded in the presence of IL-2 (50U/mL) and Akt inhibitor (Akti) (1 μM) that were supplemented every other day. Proliferative/expansion potential, phenotypical characteristics and functionality of the propagated CD19CAR T cells were analyzed in vitro and in vivo after 17-21 day ex vivo expansion. Anti-tumor activity was evaluated after adoptive transfer of the CD19CAR T cells into CD19+ tumor-bearing immunodeficient mice. Tumor signals were monitored with biophotonic imaging, and survival rates were analyzed by the end of the experiments.
We found that Akt inhibition did not compromise CD19CAR T cell proliferation and expansion in vitro, independent of the T cell subsets, as comparable CD19CAR T cell expansion was observed after culturing in the presence or absence of Akt inhibitor. Functionally, Akt inhibition did not dampen cell-mediated effector function, while Th1 cytokine production increased. With respect to phenotype, Akti-treated CD19CAR T cells expressed higher levels of CD62L and CD28 as compared to untreated CD19CAR T cells. Once adoptively transferred into CD19+ tumor-bearing mice, Akti treated CD19CAR T cells exhibited more antitumor activity than did untreated CD19CAR T cells.
Inhibition of Akt signaling during ex vivo priming and expansion gives rise to CD19CAR T cell populations that display comparatively higher antitumor activity.
KeywordsAkt inhibitor CAR T cell therapy B cell malignancies
Adoptive transfer of in vitro-expanded chimeric antigen receptor (CAR) T cells genetically modified to express tumor-targeted antigen receptors can result in dramatic regressions of B cell leukemia [1, 2]. However, insufficient persistence and effector function of CAR-redirected T cells in vivo has been a challenge for adoptive T cell therapy [3–6]. Manufacturing CAR T cells is a process that involves activation and ex vivo culture with IL-2 to facilitate CAR gene transfer and to achieve a therapeutic number of T cells. This process is also associated with decreased antitumor activity due to terminal differentiation .
Studies indicate that transfer of potent CAR T cells with central memory traits can enhance antitumor immunity and the curative potential of adoptive therapy for cancer [8–10]. Recent studies have highlighted the importance of the Akt and Wnt pathways in the regulation of CD8+ T cell differentiation and memory formation [11–14]. Upon engagement of cell surface receptors such as the T cell receptor, co-stimulatory molecules, and cytokine receptors, the PI3K-Akt pathway is activated, resulting in downstream responses via phosphorylating a range of intracellular proteins. Akt activation is required for T cell activation . However, studies have shown that the sustained activity of Akt progressively drives T cells toward terminal differentiation and diminished anti-tumor activity . Crompton and van der Waart et al. have shown that reduction of Akt activation during T cell ex vivo expansion resulted in potent tumor-infiltrating lymphocytes (TIL) and T cells specific for minor histocompatibility antigens (MiHAs) on tumors [15, 16]. We therefore hypothesized that manipulation of the magnitude of Akt activation may prevent terminal differentiation of CAR T cells as they are expanded to a therapeutic dose. To evaluate this hypothesis, we transduced and expanded CD19CAR T cells in the presence of an Akt inhibitor. We show that Akt inhibition during ex vivo expansion did not inhibit CD19CAR T cell proliferation and effector function but gave rise to less differentiated CAR T cells with high expression of CD62L and CD28 . Additionally, the CAR T cells expanded in the presence of the Akt inhibitor appeared to have enhanced antitumor activity in vivo. Taken together, these findings suggest the use of pharmacologic approaches during the ex vivo expansion of CAR T cells could achieve favorable attributes of CAR T cells for improved T cell therapy.
Unless stated otherwise, all cell lines were maintained in RPMI 1640 (Irvine Scientific) supplemented with 2 mM L-glutamine, 25 mM HEPES, and 10% heat-inactivated FCS (Hyclone). PBMCs were transformed with Epstein-Barr virus to generate lymphoblastoid cell lines (LCLs) as previously described . To generate OKT3-expressing LCLs (LCL OKT3), allogeneic LCLs were re-suspended in nucleofection solution using the Amaxa Nucleofector kit. OKT3-2A-Hygromycin_pEK plasmid was added to 5 mg/107 cells, the cells were electroporated using Amaxa Nucleofector I, and the resulting cells were grown in RPMI-1640 with 10% FCS containing 0.4 mg/mL hygromycin. To generate firefly luciferase (ffluc) + GFP+ CD19+ lymphoid leukemic cells, SupB15 cells obtained from ATCC were transduced with a lentiviral vector encoding eGFP-ffluc. Initial transduction efficiency was 50%; therefore, the GFP cells were sorted by FACS for >98% purity (fflucGFPSupB15s).
Antibodies and flow cytometry
Human T cells were analyzed by flow cytometry after staining with fluorochrome-conjugated monoclonal antibodies (mAbs) to CD8 (SKI), CD62L (Dreg-56), CD45RO (UCHL1), CD45RA (L48), CD28 (CD28.2), CD3 (SK7), IFN-γ (XMG1.2), CD107a (H4A3), KLRG1 (2 F1/KLRG1), CD57 (HNK-1), Tim3 (F38-2E2), PD1 (EH12.1), LAG3 (FAB2319C), and phosphorylated Akt (pAkt, pS473)(M89-61) (BD Biosciences). Data acquisition was performed on a MACSQuant (Miltenyi Biotec) and analyzed using FCS Express, Version 3 software (De Novo Software). Telomere length analysis was performed using the flow-FISH technique with Telomere PNA Kit/FITC obtained from Dako (Dako Denmark A/S, Denmark). Biotinylated Erbitux (cetuximab) and streptavidin-PE were used to identify T cells that expressed truncated human EGFR (EGFRt). Anti-CD4 microbeads were purchased from Stemcell Technologies, and anti-CD14, anti-CD45RA, and anti-CD25 microbeads were purchased from Miltenyi Biotec. Proliferation capacity was determined by labeling the cells with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen).
Generation of CD19 CAR T cells
For isolation of CD8+ T cells, CD4+ T cells in PBMCs were labeled with anti-CD4 microbeads and depleted with the EasySep system (Stemcell Technologies). The freshly isolated CD4 negative cells were activated with CD3/CD28 Dynabeads (Invitrogen) and transduced the next day with a lentivirus encoding CD19R (EQ):CD28:ζ/EGFRt at an MOI of 3 and expanded in the presence of 50U/mL rhIL-2 (CellGenix) and 1 μM Akt inhibitor VIII (Akti-1/2) (Merck Millipore). Cultures were then supplemented with 50 U/mL rhIL-2 (CellGenix) and Akt inhibitor every 48 h for 17–21 days before in vitro analysis and adoptive transfer. Transduced CD19CAR T cells without Akt inhibitor treatment were used as control, as were non-transduced mock cells from the same donor. In some experiments, bulk T cells, purified CD62L + CD45RO+ central memory (TCM) and CD62L+ naïve/memory T cells were engineered with CD19CAR using the selection platform that we have developed . Briefly, for TCM isolation, fresh PBMC were incubated with anti-CD14, anti-CD45RA, and anti-CD25 microbeads, and the resulting labeled cells were then immediately depleted using the CliniMACS™ depletion mode to remove CD14+ monocytes, CD25 + Treg T cells and CD45RA+ naïve T cells. The resulting unlabeled negative fraction cells were then labeled with biotinylated-anti-CD62L (DREG56) mAb and anti-biotin microbeads. The CD62L + CD45RO + TCM were purified with positive selection. Similarly, fresh PBMC were incubated with anti-CD14 and anti-CD25 microbeads, and the resulting labeled cells were then immediately depleted using the CliniMACS™ to remove CD14+ monocytes and CD25 + Treg T cells. The resulting unlabeled negative fraction cells were then labeled with biotinylated-anti-CD62L mAb and anti-biotin microbeads. The CD62L+ naïve/memory T cells were purified with positive selection.
The lentivirus CAR construct was modified from the previously described CD19-specific scFvFc:ζ chimeric immunoreceptor  to create a second-generation vector. The CD19CAR containing a CD28z costimulatory domain carries mutations at two sites (L235E; N297Q) within the CH2 region on the IgG4-Fc spacers to ensure enhanced potency and persistence after adoptive transfer . The lentiviral vector also expresses a truncated human epidermal growth factor receptor (huEGFRt), which includes a cetuximab (Erbitux) binding domain but excludes the EGF-ligand binding and cytoplasmic signaling domains. A T2A ribosome skip sequence links the codon-optimized CD19R:CD28:ζ sequence to the huEGFRt sequence, resulting in coordinate expression of both CD19R:CD28:ζ and EGFRt from a single transcript . The CD19CARCD28EGFRt DNA sequence (optimized by GeneArt) was then cloned into a self-inactivating (SIN) lentiviral vector, pHIV7 (gift from Jiing-Kuan Yee, Beckman Research Institute of City of Hope, Duarte, CA) (Additional file 1: Figure S1).
Ex vivo expanded CD8 + CD19CAR T cells with and without Akt inhibitor treatment were labeled with 0.5 mM CFSE and co-cultured with CD19+ LCLs as stimulator cells for 5 days in the presence of 50U/L rhIL-2. CFSE dilution of CD3 and CAR double positive populations was determined using multicolor flow cytometry. In some experiments, CD19CAR T cells derived from PBMC and TCM were used for proliferation assays.
Degranulation and cytokine production assays
Ex vivo expanded CD8 + CD19CAR T cells (105) with and without Akt inhibitor treatment were co-cultured with LCLs (105) in medium containing Golgi plug (BD) and antibody against CD107a for six hours at 37 °C. Acute myeloid leukemia KG1a cells were used as a negative stimulator and LCL OKT3 as positive stimulator. Degranulation was determined using multicolor flow cytometry. Ex vivo expanded CD8 + CD19CAR T cells with and without Akt inhibitor treatment (105) were co-cultured in 96-well tissue culture plates with 105 of the indicated stimulator cells. Supernatants were harvested 18 h after stimulation, and cytokines were measured by a cytometric bead array using the Bio-Plex Human Cytokine Panel (Bio-Rad Laboratories) (in triplicates) according to the manufacturer’s instructions.
To establish a leukemic model, acute lymphoid leukemic SupB15 cells were engineered with GFP firefly luciferase (GFPffluc+), and 0.5 × 106 purified GFP+ cells were inoculated into six- to ten-week old NOD/Scid IL-2RγCnull (NSG) mice intravenously (i.v) on day -5. After confirmation of the tumor engraftment, 1–2 × 106 expanded CD19CAR T cells were adoptively transferred into tumor-bearing mice intravenously. Tumor signals were monitored by Biophotonic tumor imaging.
Analyses were performed using Prism (GraphPad Software Inc.). The Mann-Whitney t- and Log-rank (Mantel-Cox) tests were used to ascertain the statistical significance of the in vivo data (tumor signals and survival). The Wilcoxon matched-pairs signed rank test (2-tailed) was used for the analysis of in vitro data except the cytokine data assumed to have a normal distribution in triplicates were analyzed with paired t test. P < 0.05 was considered statistically significant.
Akt inhibition does not compromise CD19CAR T cell proliferation and expansion
Akt inhibition generates CD19CAR T cell populations with memory-like characteristics
Akt inhibition does not dampen cell-mediated cytotoxicity but increases cytokine production
Akti-expanded CD19CAR T-cells appeared to exhibit greater anti-tumor efficacy and expansion in vivo
Effective T cell therapy against cancer is dependent on the formation of long-lived memory T cells with the ability to self-renew and differentiate into potent effector cells. These goals have been approached by various means, including using defined central memory T cells as a starting population for gene modification, optimizing CAR design, and supplementing with cytokine cocktails during ex vivo expansion [8, 18, 26, 27]. All of these strategies are feasible, but validation before translating to a clinical platform is costly and time-consuming. There are several pharmacologic candidates capable of driving T cells toward the memory phenotype, such as those activating the Wnt/β-catenin pathway  and inhibiting m-TOR mediated protein translation . However, the concentration required for significant inhibition of T cell differentiation might also lead to immunosuppressive and anti-proliferative activity [29, 30]. Our data support that addition of Akti at 1 μM during ex vivo expansion did not interfere with CD8 + CD19 CAR T cell proliferation and expansion. The same results were also observed in different T cell subsets such as bulk T cell, TCM, and naïve/memory-derived CD19CAR T cells, indicating that this is a general feature of the Akti on T cell proliferation and expansion and therefore can be used for clinical manufacturing of therapeutic doses of CAR T cell products without the restriction of starting T cell populations. Our data indicated that Akti at 1 μM resulted in modest reduction (12%) of phosphorylated Akt in the CAR T cells. A ~5-fold increase in concentration is anticipated to further reduce phosphorylated Akti at Ser 473 . However, careful titration of Akti during CAR T cell generation is necessary to maximize potency without suppressing T cell expansion and function. The optimal concentration of Akti may additionally vary among the various T cell subsets as starting population. Efficient CAR T cell therapy relies on potent effector function and in vivo persistence following adoptive transfer. Our studies demonstrated that manipulation of levels of Akt activation enhanced the antigen-specific effector function of CD19CAR T cells as indicated by elevated Th1 cytokines such as IFNγ and GM-CSF while preventing differentiation. Studies have shown that T cells with memory characteristics positively correlate with in vivo persistence and antitumor activity post adoptive transfer . Despite no statistically significant differences as a result of variation among donors and a limited number of donors, our studies consistently demonstrated that Akti-treated CD19CAR T cells exhibited higher expression levels of CD62L and CD28 without expression of exhaustion phenotypes such as KLRG1, which are the traits required for efficient anti-tumor activity following adoptive transfer . In further support, there did not appear to be a difference in telomere length between cultures with or without Akti. Upon transfer into CD19 tumor-bearing mice, Akti-treated CD8 + CD19CAR T cells efficiently inhibited tumor growth and induced long-term survival as a result of improved antitumor activity and/or expansion. The role of the Akt signaling pathway in CD4+ cells is more complicated and poorly understood than in CD8+ cells because of the interplay of regulatory versus conventional CD4+ T cells that contain different CD4+ T cell subsets. However, more tumor reduction in the mice treated with an Akti-inhibited mixture of CD4 + CD19CAR and CD8 + CD19 CAR T cells indicates the minor impact of Akti-treated Tregs on the overall therapeutic effects. This validates the use of Akti when both CD4+ and CD8+ T cells are included in the starting population for CAR T cell engineering and manufacturing. We also found that Akti-treated CAR T cells did not induce alloreactivity, an important safety consideration. Built on the pre-clinical studies [8–10, 34] demonstrating that TCM and naive/ memory T cells possess characteristics that correlate with improved anti-tumor activity, current clinical trials have developed a clinical platform for purification, transduction, and expansion of CD19CAR TCM for adoptive immunotherapy, with the goal of long-term CAR T cell disease surveillance . Our current studies demonstrate that Akti does not inhibit TCM and naïve/memory-derived CD19CAR T cell expansion but promotes the generation of memory CD19CAR T cells with greater antitumor activity and supports the use of Akti for further improved CAR T cell therapy in these advanced platforms. The underlying mechanisms may be related to changes in metabolic and transcriptional signatures characteristic of long-lived memory T cells that are reprogrammed by Akt inhibition [16, 25, 36]. Meanwhile, our data support that Akt inhibition has no impact on replicative senescence based on the lack of changes in relative telomere length and exhaustive features.
Our data suggest that inhibition of Akt signaling during ex vivo priming, expansion, and culturing gives rise to a CD19CAR T cell population that possesses increased antitumor activity. These findings suggest that therapeutic modulation of Akt might be a strategy to augment antitumor immunity for adoptive CAR T cell therapy, which could easily be transitioned into the clinic with the availability of a pharmaceutical grade Akt inhibitor.
Chimeric antigen receptor
Carboxyfluorescein succinimidyl ester
Truncated human EGFR
Green fluorescence firefly luciferase
Lymphoblastoid cell lines
- TCM :
Central memory T cells
The authors thank James Sanchez for assistance in generating the manuscripts and Saul Priceman for critical reading of the manuscript.
This study was supported by the Tim Nesvig Lymphoma Research Fund, the Skirball Foundation and two grants from the National Cancer Institute of the National Institutes of Health: the City of Hope Lymphoma SPORE (P50 CA107399) and the City of Hope Cancer Center Support Grant (P30 CA33572).
Availability of data and materials
Data are available upon request for academic researchers.
XW and SJF designed and directed the study, analyzed and organized the data, and wrote the manuscript. RU, MW, LL and CWW performed experimental work in mice and immune assays. ST and LEB reviewed the manuscript. All authors were involved with manuscript revision and all authors approved the final version.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
PBMC products were obtained from healthy consented donors under protocols approved by the City of Hope Institutional Review Board. All mouse experiments were approved by the City of Hope Institutional Animal Care and Use Committee.
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