Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Dec;19(12):2133-43.
doi: 10.1038/mt.2011.131. Epub 2011 Jul 19.

PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor

Yozo Nakazawa  1 , Leslie E Huye, Vita S Salsman, Ann M Leen, Nabil Ahmed, Lisa Rollins, Gianpietro Dotti, Stephen M Gottschalk, Matthew H Wilson, Cliona M Rooney
Affiliations

PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor

Yozo Nakazawa et al. Mol Ther. 2011 Dec.

Abstract

Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can be modified to function as heterologous tumor directed effector cells that survive longer in vivo than tumor directed T cells without virus specificity, due to chronic stimulation by viral antigens expressed during persistent infection in seropositive individuals. We evaluated the nonviral piggyBac (PB) transposon system as a platform for modifying EBV-CTLs to express a functional human epidermal growth factor receptor 2-specific chimeric antigen receptor (HER2-CAR) thereby directing virus-specific, gene modified CTLs towards HER2-positive cancer cells. Peripheral blood mononuclear cells (PBMCs) were nucleofected with transposons encoding a HER2-CAR and a truncated CD19 molecule for selection followed by specific activation and expansion of EBV-CTLs. HER2-CAR was expressed in ~40% of T cells after CD19 selection with retention of immunophenotype, polyclonality, and function. HER2-CAR-modified EBV-CTLs (HER2-CTLs) killed HER2-positive brain tumor cell lines in vitro, exhibited transient and reversible increases in HER2-CAR expression following antigen-specific stimulation, and stably expressed HER2-CAR beyond 120 days. Adoptive transfer of PB-modified HER2-CTLs resulted in tumor regression in a murine xenograft model. Our results demonstrate that PB can be used to redirect virus-specific CTLs to tumor targets, which should prolong tumor-specific T cell survival in vivo producing more efficacious immunotherapy.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Generation of human epidermal growth factor receptor 2-chimeric antigen receptor (HER2-CAR) expressing Epstein-Barr virus-specific T cells (EBV-CTLs). (a) Schema of piggyBac-transposons used to nucleofect peripheral blood mononuclear cells (PBMCs). pIRII-HER2.28.ζ and pIRII-ΔCD19 are piggyBac-transposon plasmids. pCMV-PB was the piggyBac-transposase plasmid. IR, inverted terminal repeat; CMV, cytomegalovirus immediate early promoter; IRES, internal ribosomal entry site; pA, polyadenylation sequence; HER2.28.ζ, Her2/neu-specific chimeric antigen receptor, CD28 and T cell receptor ζ chain; ΔCD19, truncated CD19. (b) Production of HER2-CTLs. On day-1, 10 × 106 PBMCs were incubated in interleukin (IL)-7 containing T-cell medium [IL-7-T-cell medium (TCM)] for 20–24 hours. On day 0, IL-7-treated PBMCs were nucleofected with pIRII-HER2.28.ζ pIRII-ΔCD19 and pCMV-PB using the Nucleofector device and immediately transferred into medium containing IL-7-TCM for 20–24 hours. On day 1, to generate EBV-CTLs, nucleofected PBMCs were stimulated with 40 Gy γ-irradiated autologous EBV-transformed B lymphoblastoid cell lines (EBV-LCLs) at a responder: stimulator ratio of 40: 1 in IL-4/IL-7-TCM. On day 9 or 10, the cells were restimulated with LCLs at a 4:1 ratio in IL-15-TCM. On day 16-17, EBV-CTLs were selected for CD19 expression using anti-CD19 MACS beads, immediately transferred in 30 ml of IL-15-TCM in a gas-permeable cell culture device (GRex) and stimulated with 5 × 106 autologous LCLs. On day 30–31, EBV-CTLs were harvested, analyzed, and cryopreserved. The total cell numbers of HER2-CAR- and ΔCD19-nucleofected EBV-CTLs were determined using trypan blue exclusion on day 1, day 9–10, day 16–17, and day 30–31. Data shows the mean ± s.d. of experiments from three donors.
Figure 2
Figure 2
Human epidermal growth factor receptor 2-chimeric antigen receptor (HER2-CAR) expression on nucleofected EBV-CTLs. HER2-CAR- and ΔCD19-nucleofected EBV-CTLs (HER2-CTLs) were analyzed for transgene expression on day 16–17 (before selection) and on day 30–31 (after selection). Nontransfected EBV-CTLs (NT CTLs) were also analyzed as controls. Shown are results from three separate donors. CTLs, cytotoxic T lymphocytes; EBV, Epstein-Barr virus; NT, nontransfected.
Figure 3
Figure 3
Immunophenotype of HER2-CTLs selected and expanded in vitro. Immunophenotyping using anti-CD4, anti-CD8, anti-CD45RO and anti-CD62L antibodies was performed after selection and expansion of HER2-CTLs in vitro. Shown are representative results from donor 2 and donor 3 of four donors. CTLs, cytotoxic T lymphocytes. CTLs, cytotoxic T lymphocytes; HER2, human epidermal growth factor receptor 2.
Figure 4
Figure 4
Antigen specificity of HER2-CTLs. After CD19-selection and 14 days-expansion in vitro, HER2-CTLs were tested for their ability to target HER2-positive tumor cell lines (Daoy and U373), a HER2-negative tumor cell line (MDA-MB-468), and autologous and allogeneic EBV-LCLs. (a) Cytotoxic specificity for Daoy, U373, MDA, autologous and allogeneic EBV-LCLs, and autologous activated T blasts using Cr51 release assay. NT EBV-CTLs were tested as controls. (b) Interferon (IFN)-γ production by HER2-CTLs in response to representative EBV antigens, EBNA1, EBNA3A, 3B, 3C, LMP1, LMP2 and BZLF1, and autologous LCLs was measured using ELISPOT assays. (c) IFN-γ production in response to HER2-positive tumor cell lines (Daoy and U373) and HER2-negative tumor cell line (MDA) using ELISPOT assay. NT EBV-CTLs were used as controls in all assays. Similar results were obtained in four donors. CTLs, cytotoxic T lymphocytes; EBV, Epstein-Barr virus; HER2, human epidermal growth factor receptor 2; LCLs, B lymphoblastoid cell lines; NT, nontransfected.
Figure 5
Figure 5
Eradication of HER2-expressing tumor cells by HER2-CTLs. On day 7 after the 4th LCL-stimulation, 1 × 106 HER2-CTLs were cocultured with 1 × 106 HER2+ U373 cells. CTLs and U373 cells were plated alone in TCM without cytokines in each well of a 24-well plate as controls. Five days later, cultures were stained with anti-CD3-FITC antibody and anti-Her2/neu-PE antibody and immediately analyzed by flow cytometry. CTLs, cytotoxic T lymphocytes; HER2, human epidermal growth factor receptor 2; LCLs, B lymphoblastoid cell lines.
Figure 6
Figure 6
Transient and reversible increase of chimeric antigen receptor (CAR) expression on HER2-CTLs by antigen-specific stimulation. On day 67 of culture, 14 days after the 5th stimulation with autologous LCLs, HER2-CTLs were stimulated with autologous LCLs, HER2-positive U373, or HER2-negative MDA cell lines. HER2-CTLs were analyzed for HER2-CAR- and ΔCD19-expression before the stimulation on day 67 of culture (day 0 prestimulation), and on day 68 (day 1 after stimulation), 70 (3), 72 (5), 74 (7) and 81 of culture (day 14 after stimulation). CTLs, cytotoxic T lymphocytes; HER2, human epidermal growth factor receptor 2; LCLs, B lymphoblastoid cell lines.
Figure 7
Figure 7
Long term survival and stable chimeric antigen receptor (CAR)-expression of HER2-CTLs in vitro. (a) HER2-CTLs received 5th LCL-stimulation at an effector: target ratio of 4: 1 on day 46 post nucleofection, and thereafter they were maintained without any antigen-stimulation in IL-15-TCM. Cells were analyzed for human epidermal growth factor receptor 2-chimeric antigen receptor (HER2-CAR) and ΔCD19 expression every 2 weeks until day 102 post nucleofection. (b,c) On day 95 post nucleofection in a, 1 × 106 cells of 5th–stimulated HER2-CTLs were further stimulated at a 4:1 ratio with autologous LCLs, or HER2 + Daoy or U373 cells or with HER2-MDA cells, or were unstimulated. Seven days later, cells were analyzed for HER2-CAR and ΔCD19 expression by flow cytometry in b and counted using trypan blue exclusion for viable cells in c. As control, NT CTLs were also stimulated with LCLs, analyzed by flow cytometry and counted as same above. CTLs, cytotoxic T lymphocytes; HER2, human epidermal growth factor receptor 2; LCLs, B lymphoblastoid cell lines; NT, nontransfected.
Figure 8
Figure 8
Adoptively transferred HER2-CTLs induce regression of HER2-positive xenografts in vivo. 5 × 104 eGFP.FFLuc-expressing HT-1080 cells were injected stereotactically into the caudate nucleus of 9–12 week old SCID mice followed by intratumoral injection of 2 × 106 HER2-CTLs or NT EBV-CTLs on day 6 after tumor inoculation. (a) Tumors grew progressively in untreated mice as shown for two representative animals (upper row) and in mice receiving NT EBV-CTLs (middle row), while tumors regressed over a period of 2–5 days in response to a single injection of HER2-CTLs generated from the same donor (lower row). (b) Quantitative bioluminescence imaging: HER2-CTLs induced tumor regression when compared to NT EBV-CTLs (two-tailed P value = 0.006, Mann–Whitney U test). Solid arrows: time of T-cell injection; open arrows: background luminescence (mean~105 photon/sec/cm2/sr); n, number of animals tested in each group. (c) Kaplan–Meier survival curve: Survival analysis performed 80 days after tumor establishment. Mice treated with HER2-CTLs had a significantly longer survival probability (P < 0.007) in comparison to untreated mice and mice that received NT EBV-CTLs. CTLs, cytotoxic T lymphocytes; HER2, human epidermal growth factor receptor 2; EPV, Epstein-Barr virus; NT, nontransfected.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Schmidt SM, Schag K, Müller MR, Weck MM, Appel S, Kanz L.et al. (2003Survivin is a shared tumor-associated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells Blood 102571–576. - PubMed
    1. Zendman AJ, Ruiter DJ., and, Van Muijen GN. Cancer/testis-associated genes: identification, expression profile, and putative function. J Cell Physiol. 2003;194:272–288. - PubMed
    1. Eshhar Z, Waks T, Gross G., and, Schindler DG. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc Natl Acad Sci USA. 1993;90:720–724. - PMC - PubMed
    1. Pulè MA, Straathof KC, Dotti G, Heslop HE, Rooney CM., and, Brenner MK. A chimeric T cell antigen receptor that augments cytokine release and supports clonal expansion of primary human T cells. Mol Ther. 2005;12:933–941. - PubMed
    1. Finney HM, Akbar AN., and, Lawson AD. Activation of resting human primary T cells with chimeric receptors: costimulation from CD28, inducible costimulator, CD134, and CD137 in series with signals from the TCR zeta chain. J Immunol. 2004;172:104–113. - PubMed

Publication types

MeSH terms