Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation
The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB). The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.
KeywordsT lymphocytes CAR manufacture gene transfer RNA electroporation
T lymphocytes can be modified by gene transfer to enhance their specific anti-tumor activities for cancer treatment (Brentjens et al., 2013; Lee et al., 2015; Morgan et al., 2006; Porter et al., 2011). To further improve this therapeutic approach, efforts are underway to define and generate better T cells. In general, T lymphocytes must be expanded to sufficient quantities before use. Several ex vivo cell manufacturing platforms can be used to produce clinical-grade products with large numbers of T cells for adoptive immunotherapy trials. These approaches include the use of anti-CD3/CD28 beads (Levine et al., 1997), the direct addition of anti-CD3 antibodies to peripheral blood mononuclear cells (PBMCs) in the presence of IL-2 (OKT3/IL-2) (Riddell and Greenberg, 1990) and cell-based artificial APCs (Suhoski et al., 2007). T cells generated by different methods have different phenotypes and in vitro/in vivo functions. The development of manufacturing strategies to generate T cells with maximal anti-tumor activities in vivo will significantly impact T-cell-based adoptive immunotherapy. All current T cell manufacturing procedures require antibodies, which are limiting factors and potential impediments due to both their cost and supply when large quantities of expanded T cells are required. Moreover, the mouse origin of the antibodies may be carried over to the T cell products, potentially rendering them immunogenic and thereby limiting the therapeutic efficacy of the infused T cells. In our previous report, a comparison of T cells generated from two methods commonly used in clinical trials showed that compared with OKT3/IL-2-stimulated T cells, CD3/CD28-Dynabead-stimulated T cells were more uniformly central memory cells with a significantly potent ability to control leukemia in Nalm6 mice model following intravenous infusion (Barrett et al., 2014). In our current study, intraperitoneal injection of mesothelin CAR RNA-electroporated T cells generated by OKT3/IL-2 stimulation achieved a rapid and sustained reduction in disease burden than those generated using CD3/CD28 Dynabead against intraperitoneal human-derived mesothelioma tumors that had grown in mice for 56 days before treatment (Campagnolo et al., 2004; Zhao et al., 2010). Furthermore, we found that T cells could be efficiently stimulated and expanded by direct electroporation of PBMCs with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment (scFv) against CD3 (OKT3) and the intracellular domains of CD28 and 4-1BB (OKT3-28BB) in the presence of IL-2. We also found that co-electroporation with other RNA molecules, such as CD86 and 4-1BBL, can further change the phenotype and function of OKT3-28BB RNA-electroporated T cells (RNA-T cells). Interestingly, T cells expanded by co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed less differentiated phenotypes, although they still maintained a tumor lytic ability as potent as that of OKT3/IL-2-stimulated T cells. In different tumor mouse models, T cells expanded from OKT3-28BB/CD86/4-1BBL RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells and similar to those of CD3/CD28 Dynabead T cells. Hence, T cells with both a young phenotype and potent killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be potentially further optimized by simply co-delivering other splices of RNA.
RNA CAR-transferred T cells expanded via OKT3/IL-2 were heterogeneous in phenotype and had enhanced and persistent function in vitro
T cells can be stimulated and expanded by electroporating mRNA encoding a chimeric membrane protein consisting of an scFv against CD3 from OKT3 and the intracellular domains of CD28 and 4-1BB
Less differentiated T cells with potent killing ability can be generated by co-electroporation of RNAs encoding OKT3-28BB and co-stimulatory molecules
Potent in vivo anti-tumor activities of RNA electroporated or lentiviral-transduced RNA-T cells
The culture of harvested lymphocytes with a soluble anti-CD3 antibody (OKT3) in the presence of interleukin (IL)-2 (Riddell and Greenberg, 1990) has been widely used and continues to be used in current cell therapy trials. Preliminary studies suggest that this method of expansion produces cells that are largely effector memory and effector T cells in phenotype (Gattinoni et al., 2005; Powell et al., 2005). Potential improvements have been explored, including the addition of feeder cells and the use of supplemental cytokines (Yang et al., 2010; Yang et al., 2013). In our current study, we sought to develop a novel T cell ex vivo expansion method that could easily be improved further. It has been reported that T cells can be stimulated and expanded by the K562 cell line expressing OKT3 scFv and other immune accessory molecules (Butler et al., 2012) and that synergy between CD28 and 4-1BB co-stimulation can be achieved by including their cytoplasmic domains arranged in tandem (Kloss et al., 2013; Stephan et al., 2007; Watts, 2005). In our study, an OKT3-28BB molecule was designed to combine stimulation (Anti-CD3 scFv is expressed on the cell surface to provide cis- and trans-stimulation to T cells) and co-stimulation (CD28 and 4-1BB, provided in cis form to T cells stimulated via anti-CD3 scFv) in a single structure for efficient human T cell expansion. It was found that T cells could only be expanded by starting with PBMCs, not with purified T cells (data not shown), suggesting that some cell components in the PBMCs essentially served as feeder cells to support efficient T cell expansion.
To develop a large number of T cells for adoptive immunotherapy, broad ex vivo cell expansion is required. Observations in murine tumor models and clinical trials have indicated that in vivo tumor treatment efficiency is mostly dependent on the differentiation status of the adoptively transferred T cells, where T cell differentiation is inversely related to in vivo anti-tumor effectiveness (Besser et al., 2010; Gattinoni et al., 2005). Thus, the use of “young”, less differentiated T cells with longer telomeres (Zhou et al., 2005b), high expression levels of CD27 and CD28 (Zhou et al., 2005a) and potent tumor lytic activities is crucial for success. In our previous work in a xenograft model of leukemia, we compared two T cell manufacturing methods that are commonly used in adoptive immunotherapy clinical trials, CD3/CD28 Dynabead and OKT3/IL-2. We found that CD3/CD28 Dynabead T cells mediated a greater anti-tumor response when mice were treated via intravenously infusion of CD19 CAR RNA-transferred T cells. This finding was consistent with our current findings that the CD19 CAR lentivirus-transduced CD3/CD28 Dynabead T cells mediate a greater anti-tumor response than do OKT3/IL-2 T cells in the same leukemia mouse model (Fig. 6E). However, compared with the CD3/CD28 Dynabead T cells, the OKT3/IL-2 T cells had a more heterogeneous phenotype and increased and sustained lytic activity in vitro. This finding suggests that the OKT3/IL-2 T cells can more potently induce tumor regression if the T cells are applied locally to the tumor sites, where less differentiated T cells with greater migration and proliferation ability are not as critically important as the T cells that are systematically infused. Upon intraperitoneally injecting T cells into mice to treat large pre-existing intraperitoneal human-derived tumors that had been growing in vivo for 56 days, we found a rapid and significant reduction in disease burden in mice receiving T cells expanded with OKT3/IL-2 (Fig. 2A and 2C). Significantly (10-fold) fewer T cells were detected in the peripheral blood of mice treated with mesothelin CAR-transferred OKT3/IL-2 T cells compared to mesothelin CAR-transferred CD3/CD28 Dynabead T cells (Fig. 2C). This result suggests that there was less leakage of OKT3/IL-2 T cells administered locally into the peripheral circulation due to their relatively high differentiation status and poor migration ability. This characteristic resulted in quick and improved local anti-tumor activities as well as the potential advantage of increased safety by avoiding unwanted off-target or on-target off-organ toxicities. Therefore, it might be important to consider the use of different T cell manufacturing procedures for treatments that require different T cell infusion routes. T cells expanded by RNA electroporation of OKT3-28BB are phenotypically and functionally similar to OKT3/IL-2 T cells and may be considered an alternative for treating cancers when local T cell administration is required. Instead of developing an alternative to pre-existing T cell manufacturing methods, the major goal of this study was to establish a stimulation and expansion platform upon which T cell quality could be easily improved to meet the required phenotypic characteristics, homing capacities, and potent effector functions for effective cancer immunotherapy (Rosenberg, 2008).
In addition to the direct use of tumor infiltrate lymphocytes (TILs) to treat cancer patients, T lymphocytes can be modified by gene transfer methods to permanently or transiently express therapeutic genes to enhance and expand their therapeutic potential (Maus et al., 2014). The requirements for transiently and permanently genetically modified T cells differ; immediate potent tumor killing ability and proper migration ability are more important for cells with transient gene transfers. By contrast, longevity, robust proliferative potential and the capacity to reconstitute the wide-ranging diversity of the T cell compartment are more critical for cells with permanent gene modifications. Extensive studies have focused on T memory stem cells (TSCM), defined as CD45RO−, CD45RA+, CD28+, CD27+, CCR7+, CD62L+, and IL-7Rα+ T cells, with increased levels of IL-2Rβ, LFA-1, CD95, and CXCR3; these studies have demonstrated several distinctive functions of memory cells. Compared with known memory populations, TSCM have shown increased proliferative capacity, greater efficiency in reconstituting immunodeficient hosts, and superior anti-tumor activities in animal models. TSCM identification is directly associated with vaccine design and T-cell-based therapies. TSCM can induce effective tumor regression when a limited number of cells are used. Tumor eradication may involve various components of the immune system. Thus, it is reasonable to transfer T cells to maintain a constant immunological attack against tumor masses. Therefore, a strategy that generates and expands TSCM-like cells is useful for the development of successful T-cell-based therapies (Gattinoni et al., 2011; Gattinoni et al., 2017). Human TSCM CAR T cells can be generated by CD3/CD28 stimulation with IL-7 and IL-15. CAR-modified CD8+ TSCM mediated superior and more durable anti-tumor responses than cells generated with protocols employed in clinical trials (Sabatino et al., 2016). One of the advantages of using RNA electroporation is that multiple splices of RNA can be introduced into T cells with high efficiency. Additionally, T cells can be genetically edited with high efficiency by electroporation of CRISPR/CAS9 (Ren et al., 2016). Thus, by co-introducing OKT3-28BB with RNA encoding other molecules, such as cytokines and co-stimulatory molecules, in combination with the CRIPSR gene editing of critical genes that regulate T cell differentiation, T cells with improved in vivo anti-tumor ability can ultimately be generated.
MATERIALS AND METHODS
Cell lines and primary human T lymphocyte cultures
The Nalm6 (DSMZ, Braunschweig, Germany), Raji (ATCC, Manassas, VA, USA), and K562 (ATCC, Manassas, VA, USA) cell lines were cultured per the suppliers’ instructions. CD19-expressing K562 cells and click beetle green (CBG)-expressing Nalm6 cells were generated as previously described (Barrett et al., 2014). SK-OV3, MCF7, MDA231, and MDA468 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as instructed. PMBCs and purified primary lymphocytes from healthy donors were provided by the University of Pennsylvania Human Immunology Core. Primary T lymphocytes were stimulated and expanded using three different methods: 1. CD3/CD28 Dynabead (Life Technologies, Grand Island, NY) were used as previously described (Barrett et al., 2011). 2. For the OKT3/IL-2 approach, the obtained PBMCs were resuspended at a concentration of 2 × 106/mL in culture medium supplemented with 50 ng/mL OKT3 and 300 IU/mL IL-2. The lymphocytes were then plated at 2 mL/well in 24-well plates (Costar). 3. For RNA-T cells, PBMCs were electroporated with OKT3-28BB RNA and re-suspended at a concentration of 2 × 106/mL in culture medium supplemented with 300 IU/mL IL-2; the cells were then cultured and maintained following the same protocol for the OKT3/IL-2 T cells. The expanded T cells were aliquoted and frozen for further use. The T cells were cryopreserved in a solution of 90% fetal calf serum and 10% dimethylsulfoxide (DMSO) at 1 × 108 cells/vial.
Construction of vectors for RNA in vitro transcription (IVT), RNA IVT and electroporation
IVT vectors for CD19-BBZ, ss1-BBZ CARs, and 4D5-BBZ were constructed as previously described (Barrett et al., 2011; Liu et al., 2015). OKT3-28BB was constructed similarly to a CAR construct without the zeta cytoplasmic region, which includes the scFv from OKT3 (VL-linker-VH); the human CD28 hinge/transmembrane region; the CD28 cytoplasmic region; and the 4-1BB cytoplasmic region (Fig. S1). DNA encoding the blinatumomab BiTE (a CD19-CD3 bi-specific antibody) was synthesized based on published sequence data from patent US7575923. Human CD86 and 4-1BB cDNA were generated using RT-PCR with RNA isolated from activated T cells; the DNA was confirmed by sequencing. All genes were cloned into a pGEM.64A-based IVT vector (Zhao et al., 2003). The IVT vector was linearized by digestion with the appropriate restriction enzyme, and the mMESSAGE mMACHINE® T7 Ultra kit (Life Technologies) was used to generate the IVT RNA according to the procedure provided with the kit. The frozen stimulated T cells were thawed and cultured overnight before electroporation. Prior to electroporation, the T cells were washed three times with OPTI-MEM and re-suspended in OPTI-MEM at a final concentration of 1–3 × 108 cells/mL. Subsequently, 0.1 mL of the T cells was mixed with the indicated IVT RNA and electroporated in a 2-mm cuvette (Harvard Apparatus BTX, Holliston, MA) using an ECM830 Electro Square Wave Porator (Harvard Apparatus BTX) (Zhao et al., 2006).
Flow cytometry analysis
Antibodies were obtained as follows: anti-human CD3 (BD Biosciences, 555335), anti-human CD8 (BD Biosciences, 555366), anti-human CD107a (BD Biosciences, 555801), and anti-human CD137 (BD Biosciences, 555956). The antibodies were incubated with T cells at 4°C for 25 min and washed twice (PBS with 2% FBS). Mesothelin CAR, ErbB2 CAR, CD19 CAR, and OKT3-28BB expression were detected by biotin-labeled polyclonal anti-mouse F(ab)2 antibody (Jackson Immunoresearch). Samples were then stained with phycoerythrin-labeled streptavidin (eBioscience, 17-4317-82). Flow cytometry acquisition was performed on either a BD FACSCalibur or Accuri C6 Cytometer (BD Biosciences). Analysis was performed using FlowJo software (Treestar).
Enzyme-linked immunosorbent assay (ELISA)
Target cells were washed and suspended at 1 × 106 cells/mL in R/10 (RPMI1640 with 10% FBS). One hundred thousand target cells of each type were added to each of 2 wells of a 96-well round bottom plate (Corning). Effector T cell cultures were washed and suspended at 1 × 106 cells/mL in R/10. One hundred thousand effector T cells were combined with target cells in the indicated wells of the 96-well plate. Additionally, wells containing only T cells were prepared. The plates were incubated at 37°C for 18 to 24 h. After incubation, the supernatant was harvested and subjected to ELISA using standard methods (Pierce, Rockford, IL).
The cells were plated at an effector:target (E:T) cell ratio of 1:1 (105 effectors:105 targets) in 160 µL of R/10 medium in a 96-well plate. An anti-CD107a antibody was added and incubated with the cells for 1 h at 37°C before Golgi Stop was added and incubated for an additional 2.5 h. The anti-CD8 and anti-CD3 antibodies were added and incubated at 37°C for 30 min. After incubation, the samples were washed once and subjected to flow cytometry using a BD FACSCalibur. The data were analyzed by FlowJo software.
Flow cytotoxic T lymphocyte (CTL) assay
Mouse xenograft studies
NSG mice were obtained from the Jackson Laboratory (Bar Harbor, ME) or bred in-house under an approved institutional animal care and use committee (IACUC) protocol and maintained under pathogen-free conditions. Six- to ten-week-old NOD-SCID-c−/− (NSG) mice were bred in-house under an approved institutional animal care and use committee protocol. For the Nalm6 leukemia model, 1 × 106 Nalm6-CBG cells (Nalm6 transduced via lentivirus with the click beetle green luciferase gene) were injected into each mouse via the tail vein. The T cells were injected via the tail vein either 5 or 7 days after the Nalm6-CBG cells were injected, as indicated. Tumor growth was monitored using bioluminescence imaging (BLI) as previously described (Barrett et al., 2011). For the M108 mesothelioma model (Carpenito et al., 2009; Zhao et al., 2010), animals received intraperitoneal injections with 8 × 106 viable M108-Luc. Tumor growth was monitored using BLI every two weeks for 4 weeks after the tumor was injected. T cells were injected intraperitoneally 8 weeks after tumor inoculation. Tumor growth was monitored using BLI every week after T cell injection. For the SK-OV3 ovarian cancer model, studies were performed as previously described with certain modifications (Liu et al., 2015). Briefly, 6- to 10-week-old NOD-SCID-γ−/− (NSG) mice were subcutaneously injected with 5 × 106 SK-OV3-CBG tumor cells in the right flank on day 0. The mice were treated with T cells via the tail vein on day 18 post-tumor inoculation, when the tumors were approximately 200 mm3 in volume.
Anesthetized mice were imaged using a Xenogen Spectrum system and Living Image v3.2 software. The mice were given an intraperitoneal injection of 10 mg/kg body weight D-luciferin (Caliper Life Sciences, Hopkinton, MA) suspended in sterile PBS at a concentration of 15 mg/mL (100 μL luciferin solution/10 g mouse body weight). Previous titration of both Nalm6 and human T cells transduced with the firefly luciferase vector revealed a time to peak of photon emission of five minutes, with peak emission lasting 6–10 min. Each animal was imaged alone (for photon quantitation) or in groups of up to 5 mice (for display purposes) in the anterior-posterior prone position at the same relative time point after luciferin injection (6 min). Data were collected until the mid-range of the linear scale was reached (600 to 60,000 counts) or until maximal exposure settings were reached (f stop 1, large binning and 120 s) and were then converted to photons/s/cm2/steradian to normalize each image for exposure time, f stop, binning and animal size. For anatomic localization, a pseudocolor map representing light intensity was superimposed over the grayscale body-surface reference image. For data display purposes, mice without luciferase-containing cells were imaged at maximal settings, and a mean value of 3.6 × 105 p/s/cm2/sr was obtained. Mice with luciferase-containing Nalm6 typically became moribund with leukemia when the photon flux approached 5 × 1011 p/s/cm2/sr, giving a detection range of 6 orders of magnitude.
Analyses were performed using STATA version 10 (StataCorp, College Station, Texas) or Prism 4 (Graphpad Software, La Jolla, CA). The in vitro data represent the mean of duplicates, and comparisons of means were performed using the Mann-Whitney test. For comparisons among multiple groups, Kruskal-Wallis analyses was performed with Dunn multiple comparison tests to compare individual groups. The leukemia burdens, as measured by BLI of the different groups, were compared with the Mann-Whitney test. The Student’s t-test was performed to compare differences in T cell proliferation.
This study was supported by research grant R01CA120409 (CHJ, YZ) and a research grant from Tmunity Therapeutics Inc. (YZ).
COMPLIANCE WITH ETHICS GUIDELINES
Xiaojun Liu, Carl H. June, and Yangbing Zhao have financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Yangbing Zhao has received research grant from Tmunity Therapeutic Inc. Shuguang Jiang, Chongyun Fang, Hua Li, Xuhua Zhang and Fuqin Zhang declare that they have no Conflicts of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. This article does not contain any studies with human subjects performed by the any of the authors.
XL., S.J., C. F., H.L., X.Z., F.Z. performed research. X.L., C.H.J., Y.Z. designed the research, and X.L., Y.Z. wrote the paper.
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