Significant progress in cancer immunology has been made in understanding the roles of tumour-reactive T-cells that can recognise and destroy malignant cells. Over the years, adoptive transfer of antigen-specific T-cells, mainly cytotoxic CD8 T-cells, has been applied as a safe and robust immunotherapeutic procedure in patients to eliminate malignant cells and extend survival without major complications ([@bib40]; [@bib44]).

Epstein--Barr virus (EBV) is associated with a broad range of malignancies that are distinguished by three distinct patterns of viral latency-related gene expression. Most successful clinical outcomes were obtained with EBV-specific cytotoxic T-cells against post-transplant lympho-proliferative disease, which expresses the complete array of EBV-latency-III antigens in transplant recipients ([@bib21]). However, EBV-positive nasopharyngeal carcinoma, Hodgkin\'s lymphoma (HL), and NK/T-cell lymphoma typically express more limited and weakly immunogenic EBV-latency-II antigens including latent membrane protein 1 (LMP1) and LMP2. Particularly, LMP1 is a transmembrane oncoprotein that mimics the tumour TNF receptor family members, capable of immortalising B-cells, and enhances cell survival by increasing bcl-2 activity ([@bib22]; [@bib37]). Supported by this, although it is sometimes of low expression or absent ([@bib27]), LMP1 has been proposed as an attractive target antigen for T-cell-based immunotherapy against EBV latency-II malignancies. Numerous reports have shown that T-cells specific for the EBV-latency-II antigens in patients are usually functionally impaired ([@bib18]; [@bib30]) or suppressed in tumour microenvironments ([@bib56]; [@bib15]) and present in low frequency ([@bib16]), but also possessing the therapeutic potency and the capacity to be expanded with EBV-latency-II antigens-loaded antigen-presenting cells (APCs) *in vitro* ([@bib51]; [@bib50]). Thus, several groups, including ours, have developed *in vitro* stimulation protocols to facilitate the generation of LMP1- and LMP2-specific T-cells and have demonstrated objective long-lasting clinical responses ([@bib3]; [@bib9]).

Despite their safety and apparent clinical effectiveness, there are significant drawbacks for *in vitro* expansion of EBV-specific T-cells, such as the relatively long manufacturing time, limited availability, and comparably low avidity of effector T-cells. Considering this, several groups have developed genetically engineered T-cells with an extrinsic antigen-specific T-cell receptor (TCR) or a chimeric antigen receptor (CAR) as an alternative approach to rapidly manufacture large numbers of potent tumour-reactive effector cells. Particularly, the clinical efficacy of TCR-engineered T-cells has been successfully demonstrated in patients with melanoma, synovial cell sarcoma, and multiple myeloma using MART1- and/or NY-ESO1-specific TCR ([@bib33]; [@bib41]; [@bib38]), similar to CD19-targeted CAR-T-cells in patients with B-cell haematologic malignancies ([@bib36]; [@bib29]). Likewise, numerous groups have attempted to develop EBV-targeting engineered T-cells, either with CAR targeting CD30 ([@bib46]) and CD70 ([@bib49]), or with extrinsic TCR specific to EBV nuclear antigen 3 ([@bib47]) and LMP2a ([@bib17]; [@bib55]; [@bib57]). However, although LMP1 is considered as an attractive target to treat EBV-positive malignancies, T-cells engineered with LMP1-specific TCR have not been developed. Here, we report the functionality and specificity of a novel murine TCR, which recognises an LMP1-derived epitope presented by HLA-A\*0201 molecules. Mainly, we demonstrate that potent EBV-LMP1-specific T-cells can be efficiently generated by TCR gene transfer and exponentially expanded *in vitro* with artificial APCs regimen, suggesting potential applications in T-cell-based immunotherapy against EBV-associated diseases, including EBV-latency-II malignancies.

Materials and methods
=====================

Mice
----

Full-length HLA-A\*0201-expressing transgenic (HLA-A2 Tg) mice (C57BL/6-Tg(HLA-A2.1)1Enge/J) and NSG mice (NOD.Cg-Prkd^cscid^ Il2rg^tm1Wjl^/SzJ) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Animal experiments were performed in accordance with our institutional animal care committee guidelines.

Cell lines
----------

K562, Jurkat, T2, and 293T cells were obtained from the American Type Culture Collection (Manassas, VA, USA). EBV-transformed B-lymphoblastoid cell lines (LCLs) were prepared with EBV B95-8 strain and HLA-A subtypes were determined with sequence-based typing. For K562-based transfectants, HLA-A\*0201, CD80, 4-1BBL, EBV-LMP1 (from EBV B95-8 strain), and firefly-luciferase cDNA were cloned into the lentiviral vector pCDH-EF1 (System Bioscience, Palo Alto, CA, USA). K562 cells were first transduced with HLA-A\*0201 (K-A2). Then, K-A2 cells were transduced with EBV-LMP1 (K-A2~LMP1~) and sequentially firefly-luciferase (K-A2~LMP1/LUC~). K-A2 cells were also transduced with human CD80 and 4-1BBL for artificial APCs (K-A2~80/4-1BBL~). Stably viable clones were isolated with limiting dilutions, and gene expression was verified by immunohistochemical analysis or flow cytometry.

Peptides and reagents
---------------------

Synthetic peptides representing CD8 T-cell epitopes WT1~126~ (RMFPNAPYL), LMP1~32~ (LLLALLFWL), LMP1~92~ (LLLIALWNL), LMP1~125~ (YLLEMLWRL), LMP1~159~ (YLQQNWWTL), LMP1~166~ (TLLVDLLWL), LMP1~167~ (LLVDLLWLL), and LMP1~173~ (WLLLFLAIL) at \>85% purity were purchased from A&A Labs (San Diego, CA, USA). Monoclonal anti-mouse CD40 (FGK45.5) was from BioXCell (West Lebanon, NH, USA). High molecular-weight Poly-IC was from InvivoGen (San Diego, CA, USA), and recombinant cytokines were from Peprotech (Rocky Hill, NJ, USA). Fluorescence-conjugated antibodies were obtained from eBioscience (San Diego, CA, USA).

Immunisation and T-cell clones
------------------------------

To generate LMP1~166~-specific CD8 T-cells, HLA-A2 Tg mice were immunised intravenously with 2 × 10^6^ dendritic cells (DCs) pulsed with 10 μg ml^−1^ LMP1~166~ for 18 h, and after 7 days, the mice received an intravenous TriVax-immunisation. TriVax consists of a mixture of 150 μg LMP1~166~, 50 μg poly-IC, and 100 μg anti-CD40 antibodies. Eight days after the booster-immunisation, intracellular IFNγ-staining was performed to measure the frequency of LMP1~166~-specific cytokine-secreting CD8 T-cells. LMP1~166~-specific T-cell cloning was carried out by following procedures with minor modification as described ([@bib7]; [@bib43]). Briefly, 1.5 × 10^6^ CD8 T-cells isolated from spleen were co-cultured with 5 × 10^5^ irradiated (6000 cGy) DCs pulsed with 5 μg ml^−1^ LMP1~166~ in 24-well plate. Seven days later, IFNγ-EliSpot assays were performed. Bulk cultured T-cells were cloned at single cells per well in U-bottom 96-well plates with 3 × 10^4^ LMP1~166~-pulsed irradiated (10 000cGy) T2 and 1 × 10^5^ irradiated (5000 cGy) splenocytes in medium containing 50 IU ml^−1^ IL-2 and 5 ng ml^−1^ IL-7. Proliferating T-cell clones were evaluated for responsiveness towards LMP1~166~ using intracellular IFNγ-staining.

Cloning of LMP1-specific, HLA-A\*0201-restricted TCR
----------------------------------------------------

Total RNA from T-cell clones was isolated using an RNeasy-mini kit (Qiagen, Valencia, CA, USA), and TCRα/β genes were amplified using SMART-RACE cDNA-amplification kit (Clontech, Mountain View, CA, USA) according to the manufacturer's instructions with primers in the constant region of mouse TCRα and TCRβ chains ([@bib7]) and sequenced. Amplified TCRα/β genes were linked using two-step overlapping-PCR with primers; TCRα-reverse; 5′-cttccacgtcgccggcctgcttcagcagggagaagttggtggcgccgctgcctctcttcactctactggaccacagcctcagcgt-3′ TCRβ-reverse; 5′-ttctccctgctgaagcaggccggcgacgtggaagaaaaccctggcccc-3′ encoding furin-sensitive spacer RVKRGSG-P2A ribosomal-skip sequence ATNFSLLKQAGDVEENPGP. The full-length modified TCR-cDNA was cloned into pCDH-EF1 and sequenced.

Production and transduction of murine TCRs to peripheral blood lymphocytes
--------------------------------------------------------------------------

The use of human material was reviewed and approved by our Institutional Review Board. 293T-cells (8 × 10^6^ cells) were seeded in a 100-mm plate. Twenty hours later, 12 μg cloned pCDH-EF1 and packaging plasmids (8 μg psPAX2, 4 μg pMD2G) were simultaneously transfected using 50 μl lipofectamine-2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Two days later, lentiviruses were harvested and titrated into 293T cells. Peripheral blood lymphocytes (PBLs) were isolated from HLA-A2-positive healthy volunteers. For pre-activation, 1 × 10^6^ PBLs were cultured with anti-CD3/CD28-coated beads (1 : 1 ratio; Invitrogen) and 300 IU ml^−1^ IL-2 for 2 days. Lentiviruses encoding murine TCR (MOI=0.5) were added to the activated 5 × 10^5^ PBLs or Jurkat cells, and centrifugation was performed at 2500 rpm for 1 h at 25 °C with 8 μg ml^−1^ polybrene (Sigma-Aldrich, St Louis, MO, USA). Peripheral blood lymphocytes prepared identically without transducing murine TCR were referred as a mock-transduced control (PBL^mock^). Two days later, PBLs were evaluated for surface expression and functional specificity of murine TCRs, and used in most experiments.

Flow cytometry analysis
-----------------------

For HLA-A2:immunoglobulin (Ig) dimer staining, Dimer-X reagent (BD Bioscience, San Diego, CA, USA) was prepared; 1 μg dimer, 5 μg LMP1~166~, and 0.5 μg β2-microglobulin (Sigma-Aldrich) at 37 °C overnight. TCR-transduced PBLs (1 × 10^6^ cells) were incubated with LMP1~166~-loaded HLA-A2:Ig dimer for 40 min, and washed/stained with anti-human CD8a, CD4, and anti-mouse IgG1 for 20 min. For murine TCRs and *in vivo* persistence, 1 × 10^6^ viable cells were stained with 0.5 μg indicated antibodies for 20 min. Fluorescence was measured using a FACS Calibur (BD Biosciences) and analyzed using FlowJo software (Tree Star, Otlen, Switzerland).

*Ex vivo* expansion and evaluation of TCR-transduced cells
----------------------------------------------------------

TCR-transduced CD8 and CD4 T-cells were isolated using MACS isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany), with \>90% purity. TCR-transduced cells (1 × 10^6^ cells) were co-cultured with 5 × 10^5^ peptide-loaded K-A2~80/4-1BBL~ with 500 IU ml^−1^ IL-2. The artificial APCs were loaded with either 5 μg ml^−1^ LMP1~166~ or WT1~126~ for 6 h and irradiated (10 000 cGy)/washed before co-culturing. For comparison, cells were incubated with anti-CD3/CD28-coated beads (1 : 1 ratio). Growing cells were split every 3--4 days and re-stimulated after a 7-day interval under the same conditions. For cytokine-secretion, 5 × 10^5^ engineered cells were co-cultured with irradiated (10 000 cGy) peptide-pulsed targets (1 : 1 ratio). After 2 days, cytokines in the supernatant were determined with an ELISA kit (eBioscience). For peptide-pulsing, target cells were incubated with 1 μg ml^−1^ peptide for 6 h at 37 °C. For antigen recognition, IFNγ-EliSpot assays were performed for peptide-pulsed T2 target cells ([@bib9]). For cytotoxicity determinations, conventional 5-h chromium-release assays were performed using various target cells.

*In vivo* therapeutic antitumour experiments
--------------------------------------------

NSG mice (6-8 weeks-old) were sublethally (300 cGy) irradiated on day -1. Each mouse intravenously received 3 × 10^6^ K-A2~LMP1/LUC~ tumour cells. Seven days later, each mouse was three times intravenously infused with 2 × 10^7^ engineered PBLs at every 2-day interval (on day 7, 9, and 11). Intraperitoneal administration of IL-2 (1000 IU per mouse) was given on days 7, 9, 11, 13, 15, and 17. Tumour growth was monitored weekly by luciferase signal of bioluminescence imaging using Xenogen *in vivo* imaging system (Caliper Life sciences, Hopkinton, MA, USA). On day 20, peripheral blood samples were analysed to assess *in vivo* persistence of infused cells.

Statistical analyses
--------------------

Statistical significance for tumour growth by bioluminescence intensity was determined using two-way ANOVA test, and survival analysis was established by Kaplan--Meier curves using log-rank tests. Results are representative of data obtained from at least two independent experiments. All analyses were performed and graphs were prepared using Prism 5.01 software (GraphPad).

Results
=======

Isolation of HLA-A2-restricted LMP1~166~-specific murine TCR from CD8 T-cell clones
-----------------------------------------------------------------------------------

First, we generated EBV-specific CD8 T-cells in HLA-A2-Tg mice with the minimal epitope LMP1~166~ (TLLVDLLWL) using a novel vaccination strategy with LMP1~166~-loaded DCs followed by a mixture of LMP1~166~ peptides, poly-IC adjuvant, and costimulatory anti-CD40 antibodies (TriVax). TriVax immunisation was highly efficient in stimulating and expanding antigen-specific CD8 T-cells that were primed with antigen-loaded DCs in mice ([@bib8]). LMP1~166~-TriVax booster-immunisation after priming with LMP1~166~-loaded DCs yielded 0.5--1.5% IFNγ-producing CD8 T-cells in spleen ([Figure 1A](#fig1){ref-type="fig"}). To generate LMP1~166~-specific T-cell clones, purified CD8 T-cells were co-cultured with LMP1~166~-loaded DCs for 7 days, and functional activity of *in vitro* stimulated T-cells was evaluated. As shown in [Figure 1B](#fig1){ref-type="fig"}, HLA-A2-restricted CD8 T-cell recognition was evident not only against peptide-pulsed T2 but also against HLA-A2-positive LCLs (LCL-A2^pos^), where higher levels of T-cell responses were observed against un-pulsed or WT1~126~-pulsed LCL-A2^pos^ than against LMP1~166~-pulsed HLA-A2-negative LCLs (LCL-A2^neg^). Subsequently, CD8 T-cells were cloned by limited dilution, and proliferative T-cell clones were examined for LMP1~166~-specific IFNγ-producing reactivity ([Figure 1C](#fig1){ref-type="fig"}). TCRα and TCRβ chains from each high level of IFNγ-secreting T-cell clones against LMP1~166~ were amplified. Since TCRs consist of heterodimers, isolated TCRα and TCRβ chains were linked with furin-spacer RVKRGSG-P2A element to express as a single open reading frame, and inserted into lentiviral vector. To examine the surface expression of murine TCRs, Jurkat cells and pre-activated PBLs were transduced with murine TCRs (named S4-1, S4-6, and S4-12). Although each TCR gene was isolated from highly antigen-responding T-cell clones, levels of transduced TCR expression varied. Above all, Jurkat cells and PBLs transduced with TCR S4-12, which comprises TRAV3D3\*02 and TRBV26\*01 ([Supplementary Figure S1](#sup1){ref-type="supplementary-material"}), exhibited more than 60% and 30% of surface expression, respectively ([Figure 1D](#fig1){ref-type="fig"}). To examine TCR functionality, engineered PBLs were co-cultured with LMP1~166~-pulsed T2 and the concentration of cytokines in the supernatant was measured. TCR S4-12-expressing PBLs secreted high levels of IL-2 against LMP1~166~-pulsed targets in comparison to PBL^mock^, which were identically prepared without transferring murine TCR ([Figure 1E](#fig1){ref-type="fig"}). Jurkat cells transduced with TCR S4-12 also exhibited comparable LMP1~166~-specific IL-2 production (data not shown). Hence, TCR S4-12 was chosen for further studies.

Functional specificities of HLA-A2-restricted LMP1~166~-specific murine TCR S4-12
---------------------------------------------------------------------------------

Subsequently, we further investigated the functional specificity of the novel murine TCR S4-12 after manipulating HLA-A2-positive PBLs to express the TCR (referred to PBL^S4-12^). Levels of TCR S4-12 surface expression in the transduced PBLs were slightly different among donors, and elicited 25--35% transduction efficiency compared to that in TCR-non-transduced PBL^mock^ (data not shown). Initially, the engineered PBL^S4-12^ were stained using LMP1~166~-loaded HLA-A2:Ig dimers, which revealed comparable results with HLA-A2 tetramer assay for the immunologic monitoring ([@bib48]; [@bib54]). The representative data presented in [Figure 2A](#fig2){ref-type="fig"} showed that PBL^S4-12^ had high levels of LMP1~166~-specific TCR-transduced CD8 and CD4 T-cells (∼9% per each), whereas no significant LMP1~166~-specific staining was found in TCR S4-6-transduced PBLs (PBL^S4-6^), that was non-functional. Apparently, PBL^S4-12^ were not stained with control peptide (WT1~126~)-loaded HLA-A2:Ig dimers ([Supplementary Figure S2](#sup1){ref-type="supplementary-material"}). To assess the specific reactivity, HLA-A2-restricted LMP1-derived peptides were pulsed onto T2 cells, and co-cultured with the engineered PBL^S4-12^. Interestingly, PBL^S4-12^ were equipped to additionally recognise target cells pulsed with LMP1~167~ (LLVDLLWLL), which is one amino acid-shifted peptide from LMP1~166~, but not to other HLA-A2-restricted LMP1-derived peptides including an irrelevant WT1~126~ ([Figure 2B](#fig2){ref-type="fig"}). Moreover, PBL^S4-12^ were also stained with LMP1~167~-loaded HLA-A2:Ig dimers, but in low frequency compared to that of LMP1~166~-loaded HLA-A2:Ig dimers ([Supplementary Figure S2](#sup1){ref-type="supplementary-material"}). Subsequently, peptides were serially diluted and pulsed onto T2 cells to evaluate the functional avidity of TCR S4-12. The engineered PBL^S4-12^ recognised LMP1~166~-pulsed targets, and secreted IL-2 in a dose-dependent manner, at a concentration as low as 1 ng ml^−1^ of LMP1~166~, whereas PBL^S4-12^ recognition against LMP1~167~ was rapidly reduced by 100 ng ml^−1^ of LMP1~167~ concentration, implying that LMP1~166~ is a real or best antigenic epitope that can be recognised by TCR S4-12. Of note, the same was not observed for an irrelevant WT1~126~ ([Figure 2C](#fig2){ref-type="fig"}). As such, antigen-specific recognition and cytolytic activity of PBL^S4-12^ were validated compared with that of PBL^S4-6^. As shown in [Figure 2D and E](#fig2){ref-type="fig"}, functional activity of PBL^S4-12^ was evident, which displayed high levels of cytotoxicity towards LMP1~166~-pulsed targets, whereas PBL^S4-6^ did not respond to the target cells.

Murine TCR S4-12 can recognise endogenous processed HLA-A2/LMP1~166~ complexes
------------------------------------------------------------------------------

One potential concern with TCR S4-12 was whether PBL^S4-12^ could directly recognise endogenous LMP1-expressing target cells, rather than exogenously peptide-provided cells. DCs transfected with LMP1-RNA were applied as a target, which enabled presentation of HLA-A2/LMP1~166~ complexes (pHLA-A2/LMP1~166~) onto the cell surface by natural antigen-processing pathways. As shown in [Figure 3A](#fig3){ref-type="fig"}, high level of PBL^S4-12^ responses were observed against LMP1-RNA-electro-transferred DCs (DC/LMP1~RNA~), similar to those with LMP1~166~-pulsed DCs (DC/LMP1~166~) that showed the presence of saturated exogenous LMP1~166~. In contrast, *in vitro* generated LMP1-specific cytotoxic T-cells that were stimulated with LMP1-RNA-transfected DCs from a same EBV-seropositive healthy donor did not respond to the DC/LMP1~166~ targets ([Supplementary Figure S3](#sup1){ref-type="supplementary-material"}), indicating that the functional reactivities of PBL^S4-12^ are due to endowing TCR S4-12, not due to endogenous activities of EBV-seropositive donor. Nonetheless, exogenous LMP1~166~-loaded LCL-A2^pos^ were recognised, resulting in production of a high level of IL-2, and were efficiently lysed by PBL^S4-12^, whereas PBL^S4-12^ revealed relatively low responsiveness towards un-loaded and WT1~126~-loaded LCL-A2^pos^ though LMP1 is known to be naturally expressed in LCLs ([Figure 3B and C](#fig3){ref-type="fig"}). However, the functional reactivities of PBL^S4-12^ towards LCL-A2^pos^ target cells were significantly reduced by HLA-A2 blocking ([Supplementary Figure S4](#sup1){ref-type="supplementary-material"}), implying that the LMP1~166~-specific recognition of PBL^S4-12^ is HLA-A2-restricted. Apparently, LCL-A2^neg^ targets were not attacked while LMP1~166~ were provided. To confirm the capability of TCR S4-12 to recognise endogenously processed pHLA-A2/LMP1~166~ on target cells, we established K562-derived stable transfectants that expressed HLA-A2 alone (K-A2) and/or together with LMP1 (K-A2~LMP1~). After selection, LMP1 expression was examined in K-A2 and K-A2~LMP1~ transfectants as well as LCL, results showed much lower level of LMP1 expression in LCL than K-A2~LMP1~ cells (data not shown). Likewise, PBL^S4-12^ were effective in recognising K-A2~LMP1~ pulsed either with or without LMP1~166~ ([Figure 4A](#fig4){ref-type="fig"}), whereas TCR-non-transduced PBL^mock^ and TCR S4-6-transduced PBL^S4-6^ did not respond to the target cells ([Supplementary Figure S5](#sup1){ref-type="supplementary-material"}). In accordance, higher cytolytic activity of PBL^S4-12^ was observed against K-A2~LMP1~ as compared to that with PBL^S4-6^ ([Figure 4B](#fig4){ref-type="fig"}). These data indicate that K-A2~LMP1~ presents pHLA-A2/LMP1~166~ on the surface through intrinsic LMP1-processing machinery.

We examined the composition of PBL^S4-12^ to clarify which subsets of T-cells have effector functions. As shown in [Figure 4C](#fig4){ref-type="fig"}, PBL^S4-12^ were mainly composed of CD8 T-cells (CD8 T^s4-12^; ∼25%) but CD4 T-cells (CD4 T^S4-12^; ∼10%) were also present, which are crucial for the persistence of transferred CD8 T-cells and long-term immunologic memory T-cell responses. Notably, purified CD4 T^S4-12^ exhibited high levels of IL-2 production similar to those with that of purified CD8 T^S4-12^ in response to LMP1~166~-loaded targets including K-A2~LMP1~ ([Figure 4D](#fig4){ref-type="fig"}), as specific HLA-A2:Ig dimer bindings were observed in CD4 T-cells as well as CD8 T-cells ([Figure 2A](#fig2){ref-type="fig"}). Likewise, we manipulated cord-blood lymphocytes with TCR S4-12, which can also be used as a potential source of effector cells since the differentiation status of TCR-engineered T-cells is a factor influencing long-term *in vivo* persistence of infused cells. The engineered cord-blood lymphocytes exhibited high level of IL-2 secretion similar to that of PBLs towards LMP1~166~-loaded and/or LMP1-expressing K-A2~LMP1~ and LCL target cells ([Supplementary Figure S6](#sup1){ref-type="supplementary-material"}). Overall, these results indicate that TCR S4-12 has high affinity for the recognition of the endogenously processed pHLA-A2/LMP1~166~ target, and that functionality of TCR S4-12 is dependent on the degree of LMP1 expression in target cells.

*In vivo* therapeutic antitumour efficacy of PBL^S4-12^
-------------------------------------------------------

Next, we assessed whether adoptive transfer of PBL^S4-12^ would offer a therapeutic benefit *in vivo* using a xenogeneic model, which was systemically engrafted with luciferase-expressing K-A2~LMP1~ (K-A2~LMP1/LUC~), and a bioluminescent imaging technique to monitor tumour growth. Mice that were intravenously engrafted with K-A2~LMP1/LUC~ underwent adoptive infusion with PBL^mock^, PBL^S4-6^, or PBL^S4-12^ on day 7 ([Figure 5A](#fig5){ref-type="fig"}). Mice treated with PBL^S4-12^ had a reduced tumour progression as compared to those with PBL^mock^ or PBL^S4-6^, drawing significantly lower bioluminescent signal by day 42 ([Figure 5B and C](#fig5){ref-type="fig"}). As such, the adoptive transfer of PBL^S4-12^ revealed significantly increased median survival of the mice by more than ⩾2 weeks ([Figure 5D](#fig5){ref-type="fig"}), and the measurement of genetically modified (mouse TCR and human CD45 double-positive) cell numbers in blood at day 20 (2 weeks after cell infusion) showed sustained high numbers of PBL^S4-12^, correlated with the observed improved antitumour effects ([Figure 5E](#fig5){ref-type="fig"}), indicating that TCR-transduced PBL^S4-12^ have potential for substantial long-term engraftment *in vivo* after adoptive transfer.

*Ex vivo* expansion of CD8 T^S4-12^ and PBL^S4-12^
--------------------------------------------------

Additionally, we investigated optimal conditions for *ex vivo* expansion conditions to obtain sufficiently high numbers of TCR-transduced cells for clinical applications, which is a prerequisite for the success of adoptive immunotherapy. For these experiments, we established K-A2-based APCs expressing co-stimulatory molecules CD80 and 4-1BBL (K-A2~80/4-1BBL~), which can increase the survival of activated T-cells. Subsequently, PBL^S4-12^ and CD8 T^S4-12^ were expanded with LMP1~166~- or WT1~126~-loaded K-A2~80/4-1BBL~ and compared with conventional anti-CD3/CD28-coated beads ([Figure 6A](#fig6){ref-type="fig"}). In this setup, stimulation with LMP1~166~-loaded K-A2~80/4-1BBL~ revealed expansion of distinct clear homogeneous populations of CD8 T^S4-12^ ([Figure 6B](#fig6){ref-type="fig"}), which resulted in higher absolute numbers of CD8 T-cells compared to those with anti-CD3/CD28-coated beads (∼400-fold versus ∼200-fold increase in PBL^S4-12^ expansion); no significant T-cell expansion was found with WT1~126~-loaded K-A2~80/4-1BBL~ ([Figure 6C](#fig6){ref-type="fig"}). The *ex vivo* expanded CD8 T-cells under all conditions for 3 weeks were mainly composed of effector-memory-like CD45RO^+^/CD62L^−^ phenotypes ([Supplementary Figure S7](#sup1){ref-type="supplementary-material"}). Moreover, antigen specificity of *ex vivo* expanded TCR-engineered cells was maintained during 28-day culture ([Figure 6D](#fig6){ref-type="fig"}), indicating that our artificial APCs can promote engineered cell proliferation with a homogeneously enriched population that maintained intact antigenic functional specificity.

Discussion
==========

EBV-specific T-cells have been successfully applied to restore EBV-specific immunity in patients with EBV-latency-III malignancies, whereas they have been in limited use for the treatment of EBV-latency-II malignancies, such as NK/T-cell lymphoma. Thus, extension of current adoptive immunotherapies toward EBV-latency-II malignancies demands more efficient immunotherapeutic strategies to generate sufficient numbers of T-cells specific to EBV-latency-II antigens, such as LMP1. Consequently, we and other have developed *ex vivo* expansion protocols capable of generating LMP1-specific T-cells using LMP1-expressing APCs that were transduced with mRNA ([@bib13]; [@bib9]) or recombinant viruses ([@bib20]). However, in the clinical realm, there are significant drawbacks for reactivation of LMP1-specific T-cells because LMP1 is toxic when expressed at high levels ([@bib24]), and the precursor frequency of LMP1-specific T-cells is very low in healthy EBV-seropositive individuals ([@bib28]). Here, we explored the functional availability of genetically modified T-cells to endow antigenic specificity towards LMP1, which is a recent approach to rapidly manufacture large numbers of potent tumour-reactive effector cells. To our knowledge, this is the first report showing that T-cells engineered with LMP1-specific TCR enable them to recognise and elicit specific cytotoxicity towards LMP1-expressing tumour cells *in vitro* and in a xenogeneic allograft model *in vivo*.

Over a considerable period of time, adoptive transfer of *ex vivo*-engineered T-cells has been successful; particularly, anti-CD19-CAR-T-cells have demonstrated objective clinical responses towards B-cell malignancies, including complete remissions ([@bib36]; [@bib29]). Despite spreading and bypassing the HLA dependency, the CAR-T-cell-based approach requires surface expression of antigens and risks development of tumour escape variants ([@bib23]; [@bib1]). In this respect, though it requires selection of patients with appropriate HLA alleles, TCR-engineered T-cells provide an effective therapeutic option for patients with CAR-T-induced tumour variants due to their high sensitivity for naturally processed antigenic peptides-HLA complexes ([@bib11]; [@bib6]). Recent clinical trials using T-cells engineered with NY-ESO-1-specific TCR have shown objective responses in patients with melanoma, synovial cell carcinoma, and multiple myeloma ([@bib41]; [@bib38]). In similar strategies to develop EBV-specific TCR-based therapies, numerous reports have shown objective EBV-specificities and *in vivo* therapeutic efficacies of genetically modified T-cells with isolated TCRs specific to HLA-A2- or HLA-A11-restricted LMP2 epitopes ([@bib17]; [@bib55]; [@bib57]).

Selection of target T-cell epitopes is critical for the development of effective TCR-based T-cell immunotherapy. Here, we focus on an HLA-A2-restricted LMP1~166~ epitope, which may be a subdominant epitope from LMP1, although it could be rather dominant in some situations. Khanna and colleagues have reported HLA-A2-restricted LMP1 epitopes ([@bib28]), and demonstrated that immunisation with recombinant viruses encoding multiple LMP1 epitopes (including LMP1~166~) induced potent T-cell responses against LMP1-expressing tumours ([@bib14]). Although potential immune defects against EBV infection are not fully understood, it is generally accepted that persistent viral infection induces inefficient anergic T-cells eliciting immune tolerance and therefore fails to eliminate viral-infected cells in patients. Moreover, clonal T-cell anergy and the related adaptive tolerance is likely to remove high avidity T-cells specific for immune-dominant epitopes to a higher extent than those for subdominant epitopes, and subdominant T-cell epitopes are detected after immunisation with vaccines lacking immunodominant peptides ([@bib42]). Thus, isolation of TCRs specific for subdominant T-cell epitopes may be effective to manipulate genetic engineering of T-cell immunity. In view of this, our results show that T-cells engineered with LMP1~166~-TCR could be specifically activated using a low concentration of peptides ([Figure 2C](#fig2){ref-type="fig"}), and these cells efficiently recognised peptide-loaded LCL-A2^pos^ and LMP1-transfected tumour cells ([Figure 4](#fig4){ref-type="fig"}). These results imply that LMP1~166~-TCR could have high avidity for antigen recognition and could be activated after a strong TCR stimulus capable of initiating a signal transduction cascade. Nonetheless, engineered cells exhibited relatively low responsiveness towards un-treated LCL-A2^pos^ targets ([Figure 3](#fig3){ref-type="fig"}), in line with previous reports that demonstrate no or low responsiveness of T-cells transduced with TCR specific to EBV and HIV antigens towards native antigen-positive cells ([@bib34]; [@bib47]) and virus-infected targets ([@bib52]). Particularly, CD28-CD3ξ domain-conjugate TCR specific to EBV antigen enhanced cytokine-secretion responses towards antigen-positive targets ([@bib47]), suggesting that properties of isolated TCRs such as signalling capacity may be responsible for TCR reactivity.

The clinical success of TCR-based therapies notwithstanding, potential concerns have been raised over the use of isolated TCR, because the pairing of transduced and endogenous TCR chains in TCR-gene-modified T-cells may induce unknown and possibly hazardous self-reactive side effects ([@bib2]; [@bib53]). To this end, several groups have explored to improve transduced TCR pairing. Murine TCRs provide an alternative source for high-affinity TCRs because the murine TCR repertoire is non-tolerant to many human antigens ([@bib7]; [@bib43]). Rosenberg and colleagues have reported that clinical trials using murine TCR-transduced cells demonstrated substantial antitumour responses ([@bib26]; [@bib35]; [@bib32]) and some patients with murine TCRs developed antibodies against TCR-variable regions with no effect on the clinical outcome ([@bib12]). Furthermore, murine-human hybrid-TCRs produced by substituting the human constant region with the murine constant region showed a higher expression of the receptor, increased cytokine secretion, and enhanced antitumour activity mediated by improved TCR pairing and CD3 stability ([@bib10]; [@bib19]). Overall, these studies support that the isolated murine LMP1~166~-TCR could be applied in clinical trials with or without further genetic modification to treat EBV latency-II malignancies.

The clinical efficacy of infused T-cells correlates with their ability to sufficiently persist *in vivo* to exhibit substantial antitumour responses. Several studies have demonstrated that *in vivo* persistence and measurable antitumour immunity depends on the differential status of effector T-cells ([@bib33]; [@bib25]; [@bib45]). In view of this, a recent study reported that cord-blood T-cells can be used as a potential source for TCR-gene transfer because most of these cells belong to naïve T-cell subsets ([@bib17]). We also observed that LMP1~166~-TCR-transduced cord-blood T-cells exhibit levels of cytokine secretion similar to that of peripheral CD8 T-cells towards LMP1-expressing targets ([Supplementary Figure S6](#sup1){ref-type="supplementary-material"}). Likewise, CD4 T-cells also play a crucial role in the persistence of transferred CD8 T-cells and generation of long-term memory T-cells, and significantly contribute to tumour prevention *in vivo* ([@bib31]). A previous report showed that TCR-engineered CD4 T-cells can confer functional specificity and subsequent antitumour immunity capable of preventing the tumour growth *in vivo* ([@bib55]). Our results also showed the generation of TCR-engineered CD4 T-cells capable of recognising LMP1-expressing tumours ([Figure 4](#fig4){ref-type="fig"}), suggesting that pHLA-A2/LMP1~166~-restricted CD4 T-cells could improve proliferation and memory development of adoptively transferred CD8 T-cells. The success of T-cell-based immunotherapy usually requires large numbers of cells (⩾10^9^) with intact effector functions. Nevertheless, long-term *ex vivo* cultured T-cells possess terminally differentiated properties, demonstrating low persistence *in vivo*. Numerous groups have developed *ex vivo* T-cell culturing protocols with anti-CD3/CD28-coated activator beads ([@bib39]; [@bib4]). Particularly, Butler and colleagues have reported anti-CD3 antibody-expressing artificial APC-based system for *in vitro* expansion of CD8 T-cells under autologous assistance of CD4 T-cell ([@bib5]). We have also developed *ex vivo* engineered T-cell expansion regimes with artificial APCs expressing HLA molecules and co-stimulatory CD80 and 4-1BBL ([Figure 6](#fig6){ref-type="fig"}). Notably, the exponentially expanded TCR-transduced T-cells could still maintain their functional specificity with a homogeneously enriched CD8 T-cell population.

In summary, we describe a novel HLA-A2-restricted TCR that specifically recognises LMP1~166~ epitope and provide the first evidence that LMP1~166~-TCR engineered T-cells allow efficient recognition to display potent cytotoxicity towards engineered LMP1-overexpressing tumour cells *in vitro* and *in vivo*. Additional studies for optimising the TCR avidity that affect the specificity of TCR-transferred T-cells could facilitate clinical applications in the treatment of EBV-associated diseases, including EBV latency-II malignancies.

This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0718), and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (NRF-2015R1A2A2A01004309).

[Supplementary Information](#sup1){ref-type="supplementary-material"} accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc)

This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 4.0 Unported License.

The authors declare no conflict of interest.

Supplementary Material {#sup1}
======================

###### 

Click here for additional data file.

###### 

Click here for additional data file.

###### 

Click here for additional data file.

###### 

Click here for additional data file.

###### 

Click here for additional data file.

###### 

Click here for additional data file.

###### 

Click here for additional data file.

![**Isolation of murine TCR specific to LMP1~166~ from CD8 T-cell clones.** HLA-A2 Tg mice (*n*=2) were immunised intravenously with 2 × 10^6^ DCs loaded with LMP1~166~ peptide for priming; 7 days later, the mice received a booster immunisation with TriVax. (**A**) Eight days after the booster, splenocytes were evaluated by intracellular IFNγ-staining after co-culturing with LMP1~166~, or without peptide (No pep). Numbers in each rectangular gate represent the percentage IFNγ-positive cells of all CD8 T-cells. (**B**) After 7 days of *in vitro* stimulation with LMP1~166~-loaded DCs, antigen-induced IFNγ-secretion of CD8 T-cells was evaluated. T2, and HLA-A2-positive and HLA-A2-negative LCLs (LCL-A2^pos^, LCL-A2^neg^, respectively) that were pulsed with LMP1~166~ or WT1~126~ were used as APCs. Peptides only without APCs (T-cells only) and un-pulsed APCs (No pep) were used as controls. Results represent the average number of spots from triplicate wells with s.d. (*bars*) of the means. These experiments of (**A**) and (**B**) were repeated thrice with similar results. (**C**) A representative T-cell clone established by limiting dilution cloning was verified for CD8 expression (*upper panel*) and antigen-specificity by intracellular IFNγ-staining (*lower panel*). (**D**) The *ex vivo-*activated PBLs and Jurkat cells were transduced with the murine TCRs isolated from LMP1~166~-specific T-cell clones (S4-1, S4-6, or S4-12). Surface expression in the transduced Jurkat cells (*upper panel*) and PBLs (*lower panel*) was examined on day-2 post-transduction. TCR-non-transduced cells (mock) were used as control. Numbers in each rectangular gate represent the percentage TCR-expressing cells of all human CD8 T-cells. (**E**) Antigen-specific functionality of isolated murine TCRs. The TCR-transduced PBLs were co-cultured with peptide-pulsed T2 cells for 2 days, and the culture supernatants were measured for IL-2 production using ELISA assay. Results represent the average amount of cytokines from two independent experiments with s.d. (*bars*). LCL=lymphoblastoid cell lines; LMP=latent membrane protein; TCR=T-cell receptor.](bjc2017475f1){#fig1}

![**Evaluation of functional specificity of murine LMP1~166~-specific TCR S4-12.** (**A**) Murine TCR S4-6 and TCR S4-12-transduced PBLs (PBL^S4-6^ and PBL^S4-12^, respectively) were stained with LMP1~166~-loaded HLA-A2:Ig dimers on day 2 post-transduction and analysed by flow cytometry. Numbers in each rectangular gate represent the percentage peptide-dimer-positive cells of all human CD8 T-cells (*upper panels*) and CD4 T-cells (*lower panels*). These experiments were repeated two times with similar results. (**B** and **C**) Assessment of peptide specificity (**B**) and T-cell avidity (**C**) of TCR-transduced PBLs. PBL^S4-12^ were co-cultured with T2 cells that were pulsed with LMP1~166~, with irrelevant peptides as indicated, or without peptide (No pep) for 2 days. IL-2-ELISA assay was performed with the culture supernatants. Results represent the average amount of cytokines from triplicate wells with s.d. (*bars*) of the means. (**D**) Antigen-induced IFNγ-secretion of PBL^S4-6^ and PBL^S4-12^ was evaluated using EliSpot assay. Results represent the average number of spots from triplicate wells with s.d. (*bars*) of the means. (**E**) Cytolytic activity of engineered PBLs against LMP1~166~-pulsed T2 cells was assessed by a ^51^Cr-release assay. Points represent the average values of cytotoxicity in different E:T ratio from triplicate wells with s.d. (*bars*) of the means. Engineered PBLs only and WT1~126~-pulsed T2 cells were used as controls. These experiments were repeated twice with similar results. LMP=latent membrane protein; PBL=peripheral blood lymphocytes.](bjc2017475f2){#fig2}

![**Antitumour activity of PBL^S4-12^.** (**A**) PBL^S4-12^ were capable of recognising LMP1-expressing DCs. PBL^S4-12^ were co-cultured with peptide-pulsed, or RNA-electro-transferred DCs as indicated for 2 days. DCs without peptide (No pep), irrelevant WT1~126~, LMP2a- and WT1-RNA were used for controls. IFNγ-ELISA assay was performed with the culture supernatants. Results represent the average amount of cytokines from triplicate wells with s.d. (*bars*) of the means. (**B** and **C**) The functional specificity (**B**) and cytolytic activity (**C**) of PBL^S4-12^ were evaluated towards HLA-A2-positive or HLA-A2-negative LCLs (LCL-A2^pos^ and LCL-A2^neg^, respectively), which were pulsed with LMP1~166,~ WT1~126~, or without peptide (No pep). (**B**) LCL-A2^pos^ were established from HLA-A2 homozygous (Homo) and/or HLA-A2 heterozygous (Hetero) donors, and three different donor-derived LCLs were used (D1, D2, and D3). T2 cells were also included. IL-2-ELISA assay was performed with the culture supernatants. Results represent the average amount of cytokines from two independent experiments with s.d. (*bars*). (**C**) PBL^S4-6^ were used as control. Points represent the average values of cytotoxicity in different E:T ratio from triplicate wells with s.d. (*bars*) of the means. These experiments were repeated twice with similar results. LCL=lymphoblastoid cell lines; LMP=latent membrane protein; PBL=peripheral blood lymphocytes.](bjc2017475f3){#fig3}

![**PBL^S4-12^ recognise endogenously processed LMP1~166~/HLA-A2 complexes on tumour cells.** (**A** and **B**) PBL^S4-12^ recognise HLA-A2 and LMP1 co-expressing K562 cells (K-A2~LMP1~). K562 expressing HLA-A2 alone (K-A2) and T2 cells without peptide (No pep) or with peptides (WT1~126~ and LMP1~166~) were also included. IL-2-ELISA assay (**A**) and ^51^Cr-release assay (**B**) were performed as in [Figure 3](#fig3){ref-type="fig"} including K-A2 and K-A2 ~LMP1~ cells. (**B**) Effector PBL^S4-6^ were used as a control. Points represent the average values of cytotoxicity in different E:T ratio from triplicate wells with s.d. (*bars*) of the means. (**C** and **D**) Functional specificity of CD8 T^S4-12^ and CD4 T^S4-12^. (**C**) Expression of TCR S4-12 in CD8 and CD4 T-cells was examined by flow cytometry. Numbers in each rectangular gate represent the percentage TCR S4-12-positive cells of all human T-cell subsets. (**D**) Antigenic specificity of purified CD8 T^S4-12^ and CD4 T^S4-12^ was evaluated as described above. (**A** and **D**) Results represent the average amount of cytokines from two independent experiments with s.d. (*bars*). These experiments were repeated twice with similar results. LCL=lymphoblastoid cell lines; LMP=latent membrane protein; PBL=peripheral blood lymphocytes; TCR=T-cell receptor.](bjc2017475f4){#fig4}

![**Adoptive transfer of PBL^S4-12^ prolongs survival of mice with LMP1-expressing tumours.** (**A**) Schematic experimental plan. Sublethally irradiated (300 cGy) NSG mice intravenously received K-A2~LMP1/LUC~ (3 × 10^6^ cells per mouse) on day 0. Engineered PBLs (2 × 10^7^ cells per mouse) were infused thrice and IL-2 was administrated six times with a 2-day interval, followed by weekly bioluminescence imaging. (**B**) Time course of *in vivo* bioluminescence imaging in representative individual mice treated with PBL^S4-6^ (*n*=9) or PBL^S4-12^ (*n*=12). A group treated with PBL^mock^ (*n*=9) was included as control. Images were adjusted to the same pseudo colour scale to show relative bioluminescence changes over time. (**C**) Tumour growth was monitored by *in vivo* bioluminescence imaging. Average radiance per mouse in *B* was shown. Points represent the average values photons in mouse with s.d. (*bars*) of the means. *P-*values were calculated using two-way ANOVA test compared to the PBL^S4-6^-infused group. (**D**) Kaplan--Mayer survival curves for all groups of mice. *P*-values were determined by log-rank tests compared to the PBL^S4-6^-infused group. (**E**) Frequency of PBLs expressing murine TCR was evaluated on day 20 with blood samples from mice in (**D**). Points represent the percentage for each group of mice with s.d. (*bars*) of the means. These experiments were repeated twice with similar results. LMP=latent membrane protein; LUC=luciferase; PBL=peripheral blood lymphocytes; TCR=T-cell receptor.](bjc2017475f5){#fig5}

![***Ex vivo* expansion of the engineered CD8 T-cells and PBLs.** (**A**) Schematic experimental plan. CD8 T^S4-12^ and PBL^S4-12^ were co-cultured with artificial K-A2~80/4-1BBL~ cells, which were pulsed with LMP1~166~ or WT1~126~, compared to anti-CD3/CD28-coated beads (1:1 ratio) with 500 IU ml^−1^ IL-2. (**B**) A representative analysis of murine TCR in *ex vivo* expanded CD8 T^S4-12^ and PBL^S4-12^ stimulated with either anti-CD3/CD28-coated beads (*upper panel*) or LMP1~166~-pulsed K-A2~80/4-1BBL~ cells (*lower panel*) for 21 days. Numbers in each rectangular gate represent the percentage TCR S4-12-positive cells of all human CD8 T-cells. (**C**) Proliferation of CD8 T^S4-12^ and PBL^S4-12^ in response to various conditions. Total numbers of murine TCR-expressing CD8 T-cells was calculated. *Points*, mean numbers of CD8 T-cell expansion over time; *bars*, s.d. Data are sum of single experiment on five donors. (**D**) Functional specificity of *ex vivo* expanded CD8 T^S4-12^ and PBL^S4-12^ was evaluated as in [Figure 3B](#fig3){ref-type="fig"}. Results represent the average amount of cytokines from two independent experiments with s.d. (*bars*). These experiments were repeated thrice with similar results. LCL=lymphoblastoid cell lines; LMP=latent membrane protein; PBL=peripheral blood lymphocytes; TCR=T-cell receptor.](bjc2017475f6){#fig6}
