Systemic treatment with CAR-engineered T cells against PSCA delays subcutaneous tumor growth and prolongs survival of mice
© Hillerdal et al.; licensee BioMed Central Ltd. 2014
Received: 13 June 2013
Accepted: 7 January 2014
Published: 18 January 2014
Adoptive transfer of T cells genetically engineered with a chimeric antigen receptor (CAR) has successfully been used to treat both chronic and acute lymphocytic leukemia as well as other hematological cancers. Experimental therapy with CAR-engineered T cells has also shown promising results on solid tumors. The prostate stem cell antigen (PSCA) is a protein expressed on the surface of prostate epithelial cells as well as in primary and metastatic prostate cancer cells and therefore a promising target for immunotherapy of prostate cancer.
We developed a third-generation CAR against PSCA including the CD28, OX-40 and CD3 ζ signaling domains. T cells were transduced with a lentivirus encoding the PSCA-CAR and evaluated for cytokine production (paired Student’s t-test), proliferation (paired Student’s t-test), CD107a expression (paired Student’s t-test) and target cell killing in vitro and tumor growth and survival in vivo (Log-rank test comparing Kaplan-Meier survival curves).
PSCA-CAR T cells exhibit specific interferon (IFN)-γ and interleukin (IL)-2 secretion and specific proliferation in response to PSCA-expressing target cells. Furthermore, the PSCA-CAR-engineered T cells efficiently kill PSCA-expressing tumor cells in vitro and systemic treatment with PSCA-CAR-engineered T cells significantly delays subcutaneous tumor growth and prolongs survival of mice.
Our data confirms that PSCA-CAR T cells may be developed for treatment of prostate cancer.
KeywordsCAR T cells PSCA Genetic engineering Prostate cancer Adoptive transfer
Adoptive transfer of ex vivo-expanded tumor infiltrating lymphocytes (TILs) has shown promising results as a treatment of human cancers . However, since it is not possible to isolate and expand TILs from all patients and tumor types, an attractive alternative technology is to isolate T cells from peripheral blood of a cancer patient and genetically engineer them with a novel T cell receptor (TCR), recognizing a tumor-associated antigen in the context of human leukocyte antigen (HLA) presentation, or a chimeric antigen receptor (CAR), recognizing a tumor-associated antigen on the surface of tumor cells. The engineered T cells are expanded and adoptively transferred back to the patient. Engineered T cells with specificities for a variety of tumor-associated antigens have been developed . The first successful attempt to treat cancer patients with TCR-engineered T cells was reported from the Surgery Branch at the National Cancer Institute in 2006, where 2 patients out of 15 (13%) demonstrated objective regression of metastatic melanoma lesions when treated with MART-1-TCR-engineered autologous T cells . CARs are artificial single chain antibody fragment (ScFv)-based receptors linked to a signaling domain for T cell activation . First-generation CARs contain the CD3 ζ chain signaling domain from the TCR complex for T cell activation, whereas second-generation CARs include also a second co-stimulatory signaling domain from CD28 , 4-1BB , OX-40  or CD27 . Third-generation CARs contain two co-stimulatory signaling domains along with the CD3 ζ chain . A successful report with complete remission of two out of three B-cell chronic lymphocytic leukemia (CLL) patients using CD19-CAR T cells was reported from University of Pennsylvania in 2011 . This was followed up by successful treatment also of B-cell acute lymphocytic leukemia (ALL) in 2013 .
Prostate cancer is one of the most common cancers in the developed world. Curative treatment is not possible when the tumor has spread beyond the prostate gland. Since the prostate is a dispensable organ, T cell immunotherapy is an attractive approach for treatment of prostate cancer as it allows for targeting of tissue-specific antigens that are also expressed on malignant prostatic cells. Prostate stem cell antigen (PSCA) is a prostate tissue-restricted antigen highly expressed on primary and metastatic prostate cancer cells . PSCA has been evaluated as a DNA vaccine in an experimental model for prostate cancer  and T cell epitopes from PSCA have been identified . Furthermore, HLA-A2-positive prostate cancer patients have been found to have circulating T cells against PSCA . Positive results have been reported in a study using a bi-specific antibody against PSCA and CD3, thereby re-directing T cells towards PSCA-expressing cells . Humanized anti-PSCA antibodies have entered clinical trials [17, 18]. Herein, we use a third-generation CAR targeting PSCA, which besides the CD3 ζ chain contains the signaling domains of CD28 and OX-40. We evaluate whether primary T cells from peripheral blood of healthy volunteers transduced with a lentiviral vector encoding the PSCA-CAR molecule are able to recognize and kill cancer cells expressing PSCA both in vitro and in vivo.
Lentivirus vector design and lentivirus production
Lentivirus for target cell modification: A number of third-generation self-inactivating lentiviral plasmids expressing two transgenes separated by the sequence for the Thosea asigna virus 2A (T2A) peptide were constructed using pGreenPuro (SBI System Biosciences, Mountain View, CA). The plasmids are denoted pBMN(TurboRFP-Luc2), pBMN(copGFP-PSCA) and pBMN(copGFP-TARP), where TurboRFP encodes turbo red fluorescent protein, Luc2 encodes codon-optimized firefly luciferase, copGFP encodes copepod green fluorescent protein, PSCA encodes the human prostate stem cell antigen and TARP encodes human T cell receptor γ-chain alternate reading frame protein.
Lentivirus for T cell engineering: An anti-PSCA CAR-expressing lentiviral plasmid, pBMN(PSCA-CAR), was generated by fusing a PSCA-recognizing single chain antibody fragment, obtained through reversed genetics  with the signaling moieties of CD28, OX-40 and CD3 ζ chain, from a plasmid obtained from M Brenner, Baylor College of Medicine, Houston, TX .
Lentiviruses were produced in HEK-293 T cells using polyethyleneimine (Sigma-Aldrich, St Louis, MO) transfection. The pBMN-based lentiviral plasmid and the packaging plasmids pLP1, pLP2 and pVSV-G (Invitrogen) were used at a ratio of 2:1:1:1. The supernatant was harvested 48 and 72 hours post-transfection, concentrated through ultracentrifugation at 75,000 × g for 90 minutes and stored at -80°C. Mock lentivirus was produced using an empty pRRL lentiviral plasmid (Addgene, Cambridge, MA).
Target cell lines
The mel526 cell line was obtained from T Boon, Ludwig Institute for Cancer Research, Brussels, Belgium and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA). Mel526-based target cells were produced through lentiviral transduction followed by sorting using a FACS Aria III sorter (BD Biosciences, Franklin Lakes, NJ). Mel526 cells co-expressing TARP, copGFP, Luc2 and turboRFP will be referred to in the text as mel526(TARP), and mel526 cells co-expressing PSCA, copGFP, Luc2 and turboRFP will be referred to as mel526(PSCA).
T cells from activated and lentivirus transducted of PBMCs
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy donors using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) and cultured in RPMI-1640 supplemented with 10% human AB serum (our own production), 2 mM L-glutamine, 10 mM HEPES, 20 μM β-mercaptoethanol and 1% penicillin/streptomycin. The PBMCs were activated with 100 ng/ml OKT-3 (Nordic Biosite, Täby, Sweden) and 100 IU/ml IL-2 (Proleukin, Novartis, Basel, Switzerland) for 2 days to selectively stimulate T cells. Activated cells were transduced with 50 μl concentrated PSCA-CAR-encoding lentivirus or Mock lentivirus for 4 hours at 37°C in the presence of 10 μg/ml protamine sulphate and 100 IU IL-2 (Sigma-Aldrich). Transduction was repeated 24 hours later and the cells were cultured and expanded for 2-4 weeks before analysis. For analysis of PSCA-CAR expression, cells were stained with biotinylated protein-L (Genscript, Piscataway, NJ) , washed 3 times with PBS containing 4% BSA, followed by labeling with phycoerythrin (PE)-conjugated streptavidin (BD Biosciences) or stained with Alexa Fluor® 647 F(ab')2 Fragment of Goat Anti-Mouse IgG (H + L) (Invitrogen) and stained with an allophycocyanin (APC)-conjugated anti-CD3 or fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibody (Nordic Biosite). Flow cytometry analysis was performed using FACSCanto II or LSRII (BD Biosciences).
IFN-γ and IL-2 ELISA
Activated and PSCA-CAR-transduced or Mock-transduced T cells (105 cells) were co-cultured with mel526(PSCA) or mel526(TARP) cells at a 1:1 ratio in 96-well plates. Supernatants were collected after 16 hours. ELISA (Mabtech, Nacka Strand, Sweden) was used to detect IFN-γ and IL-2 secretion.
Activated and PSCA-CAR-transduced or Mock-transduced T cells (105 cells) were labeled for 20 minutes at 37°C with 5 μM Cell Trace Violet (Invitrogen) in PBS and then washed with cold cell culture medium containing 10% human serum to stop the labeling reaction. Labeled PBMCs were co-cultured with irradiated (50 Gy) mel526(PSCA) or mel526(TARP) cells at a 1:1 ratio in 96-well plates for 5 days. The T cells received a low dose of IL-2 (10 IU/ml) on day 1. The labeled cells were then collected and stained with an APC-conjugated anti-CD3 antibody followed by flow cytometry analysis.
CD107a degranulation flow cytometry analysis
Activated and PSCA-CAR-transduced or Mock-transduced T cells (105 cells) were co-cultured with mel526(PSCA) or mel526(TARP) cells at a 1:1 ratio in 96-well plates for 16 hours. Cells were stained with a FITC-conjugated anti-CD107a antibody and an APC-conjugated anti-CD3 antibody followed by flow cytometry analysis.
Bioluminescence in vitrokilling assay
Activated and PSCA-CAR-transduced or Mock-transduced T cells were co-cultured with luciferase-expressing mel526(PSCA) or mel526(TARP) (15000 cells) in various effector to target cell ratios (0.4:1–50:1) in flat-bottomed 96-well plates. Co-cultures were harvested 48 hours later and analyzed for luciferase expression using Steady-Glo® Luciferase Assay System (Promega Corporation, Madison, WI), according to the manufacturer’s instruction, and the luminescence was measured in a luminometer (Wallac Victor 2 Multi-label Counter, Perkin Elmer, Waltham, MA). Luciferase activity from target cells not exposed to T cells was set as 100% cell viability (survival).
Animal model for T cell treatment
Nude NMRI mice (Harlan, Netherlands) were injected subcutaneously (hind flank) with 3 × 106 mel526(PSCA) cells. One, seven and fourteen days later the mice received intravenous injection of 1 × 107 PSCA-CAR-transduced T cells or Mock-transduced T cells. Twelve mice per group were used. The tumors were measured by caliper and tumor volume was calculated using the equation (length × width2)/2. Animals were sacrificed, when tumors reached over 1000 mm3. The Uppsala Animal Ethics Committee has approved the animal studies (ID numbers C319/9 and C195/11).
Statistics were performed using GraphPad prism software version 5.04 (La Jolla, CA, USA). Statistical analysis for IFN-γ and IL-2 secretion, cell proliferation and CD107a degranulation were performed using paired Student’s t-test. Log-rank test was used to compare survival curves created by the Kaplan-Meier method. Values of p < 0.05 were considered statistically significant.
Transduced T cells efficiently express the PSCA-CAR molecule
Peripheral blood lymphocytes isolated from healthy donors were activated for 24 hours and transduced with the PSCA-CAR-encoding lentivirus, or Mock lentivirus followed by two weeks of culture. The expression of PSCA-CAR was verified using Alexa-647 F(ab')2 Fragment of Goat Anti-Mouse IgG (H + L) (Invitrogen), which labels the heavy and light chain of mouse IgG and analyzed by flow cytometry analysis. T cells were efficiently transduced and expressed significant levels of PSCA-CAR when compared to Mock lentivirus-transduced or untransduced PBMCs (Figure 1B).
PSCA-CAR T cells specifically secrete IFN-γ and IL-2 and proliferate when exposed to target cells expressing the PSCA antigen
We first wanted to evaluate the PSCA-CAR T cells, generated from peripheral blood, against target cells in vitro. We could not make use of prostate cancer target cell lines with endogenous PSCA as they have been reported to down-regulate expression of PSCA during in vitro culture . We also screen a large number of prostate cancer cell line as well as primary prostate epithelial cells at different passages for PSCA expression by flow cytometry (Additional file 1: Methods) but were unable to detect any PSCA expression (Additional file 2: Figure S1A). Immunohistochemistry analysis has detected PSCA expression in pancreatic cancer cell lines . However, we were unable to detect PSCA expression on the surface of pancreatic cancer cell lines by flow cytometry (Additional file 2: Figure S1B). There are reports that suggest that xenografted pancreatic cancer cell lines regain PSCA expression in vivo. We therefore, transplanted two human pancreatic cancer cell lines to NMRI nude mice, excised the grafts after 3–4 weeks, made single cell suspension and examined the cell-surface expression of PSCA by flow cytometry (Additional file 1: Methods). These cell lines did not regain PSCA cell surface expression (Additional file 3: Figure S2). Therefore, we took the approach to lentivirally transduce target cells (mel526) to express the relevant antigen, PSCA, or an irrelevant control antigen, TARP. Thereby we establish a target cell line with a stable and intermediate strong PSCA expression. PSCA expression levels on mel526(PSCA) is shown in Additional file 2: Figure S1C.
PSCA-CAR T cells specifically degranulate upon specific antigen recognition and kill PSCA-expressing target cells
Systemic administration of PSCA-CAR T cells delay tumor growth and prolong survival of mice with subcutaneous PSCA-expressing tumors
PSCA is a tissue-restricted antigen highly expressed on primary and metastatic prostate cancer cells in vivo[12, 24]. It may therefore be an appropriate target for cancer immunotherapy . Fully humanized antibodies against PSCA are now in clinical trial for prostate cancer but do not lead to cure . The potential of therapeutic T cells to traffic to sites of disease, expand and persist remains a major advantage compared with antibodies. In fact, complete objective remissions have been observed for some cancer patients when autologous, engineered T cells have been used for treatment [3, 10, 11].
A number of CARs have recently been developed against PSCA [21, 23, 25, 26]. Most publications on PSCA-CAR T cells use PSCA-transfected target cell lines to show T cell activity and only one publication show reactivity against a pancreatic tumor cell line with endogenous PSCA expression . It should be noted that we did not detect PSCA expression on the surface of any prostate cancer nor pancreatic cancer cell line in vitro. Neither did we detect any PSCA expression on cultured primary prostate epithelial cells at different passages. Furthermore, we did not detect any PSCA expression on xenografted pancreatic cancer cell lines that were examined. Therefore, we were limited to use transduced target cells for PSCA-CAR T cell evaluation. We chose to use stable lentiviral transduction instead of transcient transfection, which can give unnaturally high levels of transgene expression.
There is only one report were PSCA-CAR-engineered T cells has been used in an in vivo model, in that case highly immunodeficient NSG mice with transplanted human tumors transduced to express PSCA . Significant reduction in tumor growth rate was observed when the authors transferred T cells engineered with both a CAR that provides suboptimal activation upon binding of one antigen, PSCA, and a chimeric costimulatory receptor that recognizes a second antigen, PSMA, or vice versa. The authors further showed that co-transduced T cells destroy tumors that express both antigens but do not affect tumors expressing either antigen alone .
Herein, we used a third generation CAR against PSCA and we also show significant delay in tumor growth rate and significantly prolonged survival of nude mice. However, adoptive transfer of PSCA-CAR T cells alone did not cure any tumor-bearing mice. Whole-body irradiation as a preconditioning treatment before adoptive T cell transfer together with supportive administration of IL-2 have shown significantly improved results in mice . It is therefore promising that our PSCA-CAR-engineered T cells are able to delay tumor growth in vivo without irradiation preconditioning or IL-2 support, though it may be beneficial to combine those treatments in the future for better effects. More experiments are needed to determine how long PSCA-CAR-engineered T cells persist or whether they proliferate at the tumor site. For example, T cells with longer telomeres that have high capacity to proliferate have been correlated with better prognosis for the patients receiving adoptive T cell transfer . It may therefore be important to analyze the telomeres as well the phenotype of T cells and possibly select an optimal T cell subpopulation for genetic engineering and transfer. The method of T cell activation before transduction as well as the condition for in vitro culture of engineered T cells may also affect the performance of adoptively transferred CAR T cells.
We confirm others finding that adoptive transfer of PSCA-CAR T cells is a potentially promising approach to treat prostate cancer. Although the expression of PSCA-CAR on the surface of the transduced T cells was intermediate high, almost all T cells were expressing the CAR, Figure 1B. Our experiments therefore indicate that even low level of expression of the CAR may be sufficient for T cell activation and T cell-mediated killing. Although in adoptive T cell transfer only highly reactive clones are selected (secreting more than 200 pg/ml IFN-γ after co-culture with targets), no correlation between IFN-γ secretion and persistence and efficacy of the cells in vivo has been found .
Prostate cancer has, like most cancers, an immunosuppressive tumor microenvironment  and it is important to have highly active T cells that will be able to proliferate and kill tumors also in this harsh environment. Therefore, our future focus will be on enhancing the resistance of the PSCA-CAR T cells to immunosuppressive factors.
Mohanraj Ramachandran and Justyna Leja shared authorship.
Chimeric antigen receptor
Prostate stem cell antigen
Cluster of differentiation
- IgG (H + L):
Immunoglobulin heavy and light chain
Tumor infiltrating lymphocyte
T cell receptor
Human leukocyte antigen
Single chain antibody fragment
Chronic lymphocytic leukemia
Acute lymphocytic leukemia
Thosea asigna virus 2A
Copepod green fluorescent protein
T cell receptor γ-chain alternate reading frame protein
Spleen-focus forming virus
Lysosomal-associated membrane protein.
The authors wish to thank Berith Nilsson, Di Yu, Chuan Jin, Fredrik Eriksson and Angelica Loskog for technical assistance and advices and the BioVis core facility at Uppsala University for cell sorting.
The Swedish Cancer Society (12 0569), Gunnar Nilsson’s Cancer Foundation, the Swedish Children Cancer Foundation (PROJ10/027) and the Marcus and Marianne Wallenberg’s Foundation supported this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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