Telomerase (TERT) is overexpressed in 80% to 90% of primary tumors and contributes to sustaining the transformed phenotype. The identification of several TERT epitopes in tumor cells has elevated the status of TERT as a potential universal target for selective and broad adoptive immunotherapy. TERT-specific cytotoxic T lymphocytes (CTL) have been detected in the peripheral blood of B-cell chronic lymphocytic leukemia (B-CLL) patients, but display low functional avidity, which limits their clinical utility in adoptive cell transfer approaches. To overcome this key obstacle hindering effective immunotherapy, we isolated an HLA-A2–restricted T-cell receptor (TCR) with high avidity for human TERT from vaccinated HLA-A*0201 transgenic mice. Using several relevant humanized mouse models, we demonstrate that TCR-transduced T cells were able to control human B-CLL progression in vivo and limited tumor growth in several human, solid transplantable cancers. TERT-based adoptive immunotherapy selectively eliminated tumor cells, failed to trigger a self–MHC-restricted fratricide of T cells, and was associated with toxicity against mature granulocytes, but not toward human hematopoietic progenitors in humanized immune reconstituted mice. These data support the feasibility of TERT-based adoptive immunotherapy in clinical oncology, highlighting, for the first time, the possibility of utilizing a high-avidity TCR specific for human TERT. Cancer Res; 76(9); 2540–51. ©2016 AACR.

The development of adoptive cell therapy (ACT) represents an emerging and realistic approach to treat cancer patients. This is testified by the numerous phase II clinical trials, the approval of specific T-cell therapies by the FDA, and the growing interest of biotechnology and pharmaceutical industry to generate “off-the-shelf” reagents to treat a large spectrum of tumors (1). At present, three types of ACT protocols can be defined based on isolated tumor-infiltrating T cells (TIL), T-cell receptor (TCR) engineered T cells, or chimeric antigen receptor (CAR) transduced lymphocytes, all of them already tested in clinical settings (2). TIL-based ACT can result in a long-lasting and complete cancer regression in metastatic melanoma patients (3, 4). However, this approach remains a personalized treatment that displays several technical constraints (5). The clinical response following adoptive TIL transfer was associated with T cells reactive toward mutated epitopes that were able to persist in patients for at least 1 month after lymphocyte infusion (6). These boundaries intrinsic to TIL-based ACT could be surmounted by gene therapy strategies based on genetically engineered lymphocytes where the desired TCR sequence insertion, by a virus-mediated delivery into naïve T cells, can confer an antigen-oriented immune specificity (7–9). To develop rapidly and apply ACT to a wide range of human neoplastic diseases, the characterization of high-avidity TCRs that efficiently and broadly recognize cancer cells is thus a primary goal (10). However, antitumor CTLs with a high-avidity TCR against non-mutated tumor-associated antigens (TAA) are normally deleted during thymus education of self-reactive T cells (11), and isolation of TCR recognizing individual mutations of patients' cancers is feasible in theory (12) but currently not applicable to large scale, standardized therapy. Nowadays, high-avidity TCR sequences could be achieved by different approaches. T cells with higher functional avidity could be generated in vitro by stimulation with autologous dendritic cells (DC) transfected with RNA encoding an allogeneic major histocompatibility complex (MHC) and the desired TAA (13). Alternatively, TCR can be isolated from mouse CTLs primed in vivo by vaccination of transgenic mice bearing human HLA-A2 molecules (14, 15), an approach that was recently improved by the immunization of human antigen–negative mice engineered to bear the whole human TCR-α and β gene loci together with the HLA-A2 allele (16). We previously reported the feasibility to isolate and enrich a polyclonal T-cell population specific for human telomerase (hTERT)865–873 epitope through in vitro stimulation of mouse T lymphocytes isolated from HLA-A2.1 transgenic mice (17). These CTLs recognized different hTERT-expressing human cancer cell lines, as well as colon cancer stem cells (17). Telomerase is reactivated in the majority of human tumors independently of their histology (18), and several hTERT epitopes, which are naturally processed and presented in association with MHC molecules on tumor cell surface, have been already documented (19–22). It is thus not surprising that TERT was ranked among the most prioritized TAAs (23), and several active immunotherapeutic approaches based on TERT antigen have been exploited to target, both in vivo and in vitro, either autologous or allogeneic antigen-presenting cells (APC), including antigenic peptides (24–27), RNA-based vaccines (28), as well as plasmid or viral vectors encoding hTERT (29). Unfortunately, clinical responses in these trials were limited, suggesting the need for more powerful, immune-based strategies. In the current study, we show the feasibility to transduce human T cells with a high-avidity mouse TCR able to recognize hTERT865–873 peptide in association with HLA-A2 molecules to control human solid tumors and hematologic malignancies, such as chronic lymphocytic leukemia (B-CLL). The high levels of hTERT in leukemic B cells correlated with poor clinic outcome (30, 31). We show here that hTERT-based ACT can selectively eliminate leukemic B cells, causing a minor toxicity against normal myeloid cells, making this approach suitable to clinical translation.

Mice

C57BL/6 (C57BL/6NCrl) mice were purchased from Charles River Laboratories Inc.; OT-1 (C57Bl/6-Tg(TcraTcrb)1100Mjb/J) and CD45.1+ mice (B6.SJL-PtrcaPepcb/BoyJ) from The Jackson Laboratory; NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac) and Rag2−/−c−/− mice (B10;B6-Rag2tm1Fwa II2rgtm1Wjl) from Taconic. The transgenic mice IgH.TEμ have been described previously (32), and their natural history of malignant progression has been monitored (Supplementary Fig. S1) in about 150 mice. All animal experiments were approved by the Verona University Ethical Committee, authorized by Ministerial Decree (16/2014-B) and conducted according to the guidelines of the Federation of European Laboratory Animal Science Associations.

Cell lines and patient samples

All cell lines were obtained from ATCC and were passaged for fewer than 6 months after their purchase. Human cell line identities were verified using short tandem repeat profiling. Healthy donor (HD) CD34+ cells derived from bone marrow (BM) were purchased from Lonza. Peripheral blood mononuclear cells (PBMC) isolated from B-cell chronic lymphocytic leukemia (B-CLL) patients and HDs were collected at the Hematology Unit, Azienda Ospedaliera Universitaria Integrata (AOUI) in Verona (Italy). All participating persons provided written informed consent in compliance with the Declaration of Helsinki. The study was approved by the local ethics committee (AOUI of Verona, n. 1496). Selected B-CLL patients had heterogeneous Binet clinical stages (33).

Generation of hTERT865–873– and hHCV1406–1415–specific, TCR-transduced T cells

OKT-3–activated PBMCs were infected with the viral supernatant of the hTERT865–873/PG13 cell line in the presence of hIL15 (100 μg/mL) and rIL2 (300 IU/mL; ref. 34). PBMCs were then immunomagnetically enriched for CD34 (Miltenyi) and expanded. Control HCV1406–1415 (KLVALGINAV)–specific, TCR-transduced T cells were generated following the same protocol. The percentage of CD4+ and CD8+ T cells (usually 20% and 80%, respectively) was always tested before in vitro or in vivo studies. In general, the amount of T cells used for in vivo treatments were adjusted in order to inject 2.5 × 106 CD8+ cells.

Generation and expansion of telomerase-specific T cells from B-CLL and HD PBMCs

B-CLL patients and HD were selected for HLA-A2 status, as assessed by FACS. T cells were immunomagnetically isolated from PBMCs. Human DCs were generated from CD14-selected monocytes (Miltenyi Biotec) and, after 100 μg/mL LPS maturation, pulsed with 10 μg/mL of the specific peptide. DCs were then used to stimulate T cells at an E/T ratio of 10:1 in complete RPMI-1640 in presence of IL7 (10 ng/mL), IL15 (2 ng/mL), and IL2 (10 IU/mL; all from Miltenyi Biotec). At days 7 and 15 of culture, T cells were restimulated with peptide-pulsed DCs. CD8+ T cells were screened for hTERT865–873 dextramer positivity and hTERT865–873–specific reactivity in IFNγ ELISA.

Systemic treatment of mouse leukemic chimeras

Chimeras were generated combining 106 CD45.2+ IgH.TEμ and 4 × 106 CD45.1+ syngeneic WT BM cells in Rag2−/−γc−/− mice, after preconditioning with Busulfan (25 mg/kg). When CD45.2+ cells raised to 15% of total B cells, 5 × 106 mTERT198–205– or OVA257–264–specific CTLs were intravenously injected twice in mice after γ-irradiation, followed by recombinant IL2 administration (17).

Systemic treatment of human leukemic chimeras

NOG mice were γ-irradiated (1.20 Gy) and subsequently engrafted with 105 human HLA-A2CD34+ cells via tail-vein injection, as previously reported (35). Mice were then injected with 108 freshly isolated B-CLL PBMCs into retro-orbital plexus. Treatments with 2.5 × 106 hTERT865–873– or hHCV1406–1415–specific, TCR-transduced T lymphocytes were given 3 times, weekly, starting 1 week after the engraftment of the pathology and were always followed by IL2 administration. At sacrifice, organs were collected and analyzed by flow cytometry and IHC.

Statistical analysis

Data were indicated as the mean±SD. The Student t test was used to determine statistically significant differences between two treatment groups, while the ANOVA test was used in case of multiple comparisons. Growth curves were analyzed with repeated-measures (RM) ANOVA. Survival analysis was performed using the Kaplan–Meier survival analysis (log-rank) method. All P values less than 0.05 were considered statistically significant.

mTERT198–205–specific CTLs control mouse B-CLL progression

After repeated ACTs with polyclonal mouse (m)TERT198–205–specific CTLs to treat prostate cancers, we did not detect major side effects toward normal cells and tissues with the exception of a transient and reversible B-cell depletion in lymphoid organs (17). We therefore verified the therapeutic efficacy of mTERT198–205–specific CTL administration to restrain the expansion of monoclonal B cells, using the IgH.TEμ mouse model in which the sporadic SV40 large T antigen (hereafter indicated as SV40+) expression in mature B cells generates a B-CLL–like neoplasia (32). We considered mice as a leukemic (defined henceforward as IgH.TEμ+) when the majority of CD19+ B cells expressed the IgMb allele (IgMb > 75%). Splenic B cells isolated from IgH.TEμ+ mice displayed higher levels of TERT protein (Fig. 1A, top), as reported for human B-CLL (31), together with an increased TERT enzymatic activity (Fig. 1A, bottom) in comparison with B lymphocytes purified from either IgH.TEμ or WT mice. Only B cells purified from IgH.TEμ+ mice were efficiently recognized by polyclonal mTERT198–205 CTLs, both in a cytofluorimetric cytotoxic assay (summarized in Supplementary Fig. S2A) and IFNγ release assay (Fig. 1B). These findings demonstrate that leukemic B lymphocytes can naturally process the endogenous TERT198–205 peptide and present it in a MHC class I–restricted fashion.

Figure 1.

Malignant B-CLL cells isolated from IgH.TEμ+ splenocytes express high levels of telomerase and are recognized by mTERT198–205–specific CTLs. A, B cells immunomagnetically isolated from splenocytes of positive or negative transgenic and WT mice were tested for TERT expression by Western blot (top) and TERT activity by TRAP assay (bottom; normal samples, black; heat inactivated samples, gray; WT, n = 6; IgH.TEμ, n = 20; IgH.TEμ+, n = 10). Data show representative samples. B, cell killing activity of mTERT198–205–specific CTLs was evaluated by flow cytometry cytotoxic assay (top) and IFNγ secretion assay (top); positive control, mTERT198–205–pulsed, WT B cells (CTRL+: n = 6); negative control, OVA257–264–pulsed WT B cells (CTRL−, n = 6); WT B cells (n = 6); IgH.TEμ B cells (n = 20); IgH.TEμ+ B cells (n = 10). ANOVA test.

Figure 1.

Malignant B-CLL cells isolated from IgH.TEμ+ splenocytes express high levels of telomerase and are recognized by mTERT198–205–specific CTLs. A, B cells immunomagnetically isolated from splenocytes of positive or negative transgenic and WT mice were tested for TERT expression by Western blot (top) and TERT activity by TRAP assay (bottom; normal samples, black; heat inactivated samples, gray; WT, n = 6; IgH.TEμ, n = 20; IgH.TEμ+, n = 10). Data show representative samples. B, cell killing activity of mTERT198–205–specific CTLs was evaluated by flow cytometry cytotoxic assay (top) and IFNγ secretion assay (top); positive control, mTERT198–205–pulsed, WT B cells (CTRL+: n = 6); negative control, OVA257–264–pulsed WT B cells (CTRL−, n = 6); WT B cells (n = 6); IgH.TEμ B cells (n = 20); IgH.TEμ+ B cells (n = 10). ANOVA test.

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The low incidence of leukemia did not allow us to verify in vivo the therapeutic efficacy of a TERT-based ACT in IgH.TEμ mice, so we engrafted BM cells isolated from an IgH.TEμ+ mouse into immunodeficient Rag-2−/−γc−/− mice, partially ablated with chemotherapy, to establish a standardized B-CLL–like pathology. In fact, all engrafted mice developed a B-CLL with the same features of the IgH.TEμ+ mouse donor and tumor-bearing mice had to be euthanized 24 days after BM cell transplant (results for 2 passages out of 9 total are shown). The transplants did not affect the leukemic phenotype: the histological structure of the spleen was almost completely replaced by a uniform infiltration with SV40+CD19+CD5+B220low/− cells, similar to what occurred in the donor IgH.TEμ+ mouse (Supplementary Fig. S3A). Overall, B cells isolated from tumor-bearing chimeric mice showed high levels of mTERT expression and activity (Supplementary Fig. S3B) and were recognized by mTERT198–205–specific CTLs (Supplementary Fig. S3C). Therefore, the pre-leukemic B cells in BM were able to give rise to a full B-CLL–like disease.

In order to assess the in vivo efficacy of mTERT198–205–specific CTLs in immune-competent mice, we generated mouse chimeras by transplanting a mixture of BM cells, comprising one-fifth of leukemic cells (CD45.2+) derived from IgH.TEμ+ mouse (as previously described) and four-fifths of normal congenic BM-cells (CD45.1+) isolated from WT mice, into immunodeficient Rag-2−/−γc−/− mice. When mice had about 15% of CD19+ circulating cells, they were treated with two repeated transfers of either mTERT198–205– or the control, OVA257–264–specific CTLs (Supplementary Fig. S3D). The leukemic mice treated with mTERT198–205–specific CTLs displayed a significant reduction in the total number of blood circulating CD19+ B lymphocytes compared with control mice (Fig. 2A). However, mTERT-based ACT did not affect the normal B-cell reconstitution, because the levels of CD19+ B-cells in treated chimeras engrafted with only normal (WT) BM cells were not significantly different compared with untreated mice (Fig. 2A). In fact, mTERT-based ACT selectively reduced the CD19+CD45.2+ leukemic cell expansion without influencing the normal CD19+CD45.1+ B-cell development (Fig. 2B). This therapeutic effect of mTERT-specific ACT was mirrored by the significant inverse correlation between the frequencies of blood circulating, mTERT198–205–specific CTLs with the percentage of circulating B-CLL cells (Fig. 2C). The mTERT-specific ACT promoted a significant contraction in splenic SV40+ cells compared with control ACT, which was associated with an increased number of normal B220+ cells (Fig. 2D). Flow cytometry analysis on BM and spleen confirmed that, even if the overall percentage of splenic CD19+ cells was not modified by mTERT-specific ACT, the relative ratio between malignant CD45.2+ cells and healthy CD45.1+ cells significantly changed (Fig. 2E). As expected from this anti-leukemic activity, mTERT-specific ACT significantly improved the leukemia-bearing mouse survival (Fig. 2F).

Figure 2.

mTERT-based ACT controls mouse B-CLL progression. Leukemia-bearing chimeras were γ-irradiated (200 cGy) and treated with two weekly ACTs based on mTERT198–205–specific (n = 32) or OVA257–264–specific (n = 32) CTL i.v. infusions. Mice engrafted with only normal CD45.1 BM cells isolated from WT mice were either treated or not with mTERT-specific ACT (white and black triangles, respectively). A, flow cytometric evaluation of total circulating CD19+ B cells (mTERT vs. OVA ACT, P = 0.002; WT + mTERT ACT vs. WT P = 0.8; RM ANOVA test). B, flow cytometric evaluation of both circulating malignant CD19+CD45.2+ B cells (mTERT vs. OVA ACT, P = 0.013; RM ANOVA test, left) and normal CD19+CD45.1+ B cells (mTERT vs. OVA ACT, P = 0.14; RM ANOVA test, right). C, Spearman rank correlation between circulating, infused antigen-specific CTLs (anti-Vβ5.2 for OVA257–264–specific CTLs and anti-Vβ11 for mTERT198–205–specific CTLs, red and blue dots, respectively) and malignant B cells. D, distribution of B220+ cells (blue cells) and SV40+ cells (red cells) in the spleen of mice treated with either mTERT-base or OVA-based ACT. Bars, 20 μm. Data are mean ± SD of 1 of 4 independent experiments (n = 8 each). Student t test. E, flow cytometry analysis of spleen and BM in ACT-treated chimeric mice. Bar charts denote the total percentage of CD19+ cells divided into leukemic (gray bar) or WT (black bar) cells (left). Representative dot plots are shown (right). Student t test. F, ACT-treated mouse survival analysis. Mice were euthanized when the percentages of circulating malignant CD19+CD45.2+ cells were ∼80% of total PBMCs. Kaplan–Meier analysis: mTERT ACT (n = 32) versus OVA ACT (n = 32), P < 0.001.

Figure 2.

mTERT-based ACT controls mouse B-CLL progression. Leukemia-bearing chimeras were γ-irradiated (200 cGy) and treated with two weekly ACTs based on mTERT198–205–specific (n = 32) or OVA257–264–specific (n = 32) CTL i.v. infusions. Mice engrafted with only normal CD45.1 BM cells isolated from WT mice were either treated or not with mTERT-specific ACT (white and black triangles, respectively). A, flow cytometric evaluation of total circulating CD19+ B cells (mTERT vs. OVA ACT, P = 0.002; WT + mTERT ACT vs. WT P = 0.8; RM ANOVA test). B, flow cytometric evaluation of both circulating malignant CD19+CD45.2+ B cells (mTERT vs. OVA ACT, P = 0.013; RM ANOVA test, left) and normal CD19+CD45.1+ B cells (mTERT vs. OVA ACT, P = 0.14; RM ANOVA test, right). C, Spearman rank correlation between circulating, infused antigen-specific CTLs (anti-Vβ5.2 for OVA257–264–specific CTLs and anti-Vβ11 for mTERT198–205–specific CTLs, red and blue dots, respectively) and malignant B cells. D, distribution of B220+ cells (blue cells) and SV40+ cells (red cells) in the spleen of mice treated with either mTERT-base or OVA-based ACT. Bars, 20 μm. Data are mean ± SD of 1 of 4 independent experiments (n = 8 each). Student t test. E, flow cytometry analysis of spleen and BM in ACT-treated chimeric mice. Bar charts denote the total percentage of CD19+ cells divided into leukemic (gray bar) or WT (black bar) cells (left). Representative dot plots are shown (right). Student t test. F, ACT-treated mouse survival analysis. Mice were euthanized when the percentages of circulating malignant CD19+CD45.2+ cells were ∼80% of total PBMCs. Kaplan–Meier analysis: mTERT ACT (n = 32) versus OVA ACT (n = 32), P < 0.001.

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Generation and characterization of engineered hTERT865–873–specific T cells

To verify the presence of a TERT-specific immune response induced by B-CLL progression, we selected B-CLL patients, sharing HLA-A2 allele and clinical stage (according to the Binet staging system), with at least 4-year follow-up; as a control, we included HLA-A2+ HDs. We isolated CD3+ T cells from patients and HD PBMCs and cocultured them with either hTERT865–873– or hCMV495–503–peptide pulsed (as positive control of stimulation), in vitro differentiated, HLA-A2–matched DCs. After 3 weekly in vitro stimulations, we verified the presence of hTERT-specific CD8+ T cells, defined as hTERT865–873 dextramer–positive cells, in approximately 50% of the B-CLL patients but not in HDs, even though in almost all patients and HDs we detected the presence of hCMV-specific CD8+ T cells (Fig. 3A). Patients whose T cells displayed positivity for hTERT-specific dextramer staining were also able to release IFNγ in response to hTERT865–873 peptide (Fig. 3B), with a linear relationship between cytokine production and the number of antigen-specific cells in culture (Fig. 3B, inset). Then, we examined the association between the presence of endogenous anti-hTERT865–873 response and disease progression, measured as time from diagnosis to initial therapy (time to first treatment, TTFT). Comparison between TTFT curves showed a trend to a shorter TTFT in the “TERT nonresponder” group (Fig. 3C). These data clearly unveiled the previously unknown presence of endogenous T cells specific for a physiologically processed hTERT865–873 epitope in B-CLL patients.

Figure 3.

Endogenous and genetically targeted T-cell responses against hTERT. A, evaluation of endogenous anti-TERT () and anti-CMV () immune response in T cells isolated from 16 B-CLL patients and 4 age-matched HDs after in vitro stimulation with human DCs pulsed with either hTERT865–873 or hCMV495–503 peptide. Patients were divided into “TERT responders” (n = 8) and “TERT nonresponders” (n = 8) according to a hTERT865–873 dextramer positivity greater than 0.02%. ANOVA test. Representative dot plots are shown. B, IFNγ production after in vitro incubation of T cells isolated from either B-CLL patients or HDs. Columns show hIFNγ released in the presence of cells pulsed with hTERT865–873 peptide after subtraction of values obtained in the presence of control peptide-pulsed cells. Spearman rank correlation between dextramer positivity and hIFNγ release for “TERT responders” patients (inset). C, Kaplan–Meier analysis of “TERT responders” and “TERT nonresponders” patients analyzed in relation to the time (months) to first treatment. D, summary of mouse hTERT865–873–specific TCR sequence used to engineer human T cells. E, flow-cytometric analysis of mouse Vβ3 chain expression and dextramer positivity of hTERT865–873– and HCV1406–1415–specific, TCR-engineered T cells. F, functional avidity evaluation of the endogenous, HLA-A2–restricted hTERT-specific T cells isolated from two representative “TERT responders” patients (blue lines) and the hTERT865–873–specific, TCR-engineered T cells (black line) after coculture in the presence of T2 cells loaded with varying hTERT865–873 peptide concentrations. Values were normalized to IFNγ released by the “TERT responder” patient with highest response. Red dashed line, the 50% of maximum response. G, analysis of fratricidal activity of hTERT865–873–specific, TCR-engineered T cells against either PBMCs or HCV1406–1415–TCR-transduced T cells pulsed with hTERT865–873 peptide, hCMV495–503 peptide, or left unpulsed. Data are mean ± SD of 1 of 3 independent experiments. ANOVA test.

Figure 3.

Endogenous and genetically targeted T-cell responses against hTERT. A, evaluation of endogenous anti-TERT () and anti-CMV () immune response in T cells isolated from 16 B-CLL patients and 4 age-matched HDs after in vitro stimulation with human DCs pulsed with either hTERT865–873 or hCMV495–503 peptide. Patients were divided into “TERT responders” (n = 8) and “TERT nonresponders” (n = 8) according to a hTERT865–873 dextramer positivity greater than 0.02%. ANOVA test. Representative dot plots are shown. B, IFNγ production after in vitro incubation of T cells isolated from either B-CLL patients or HDs. Columns show hIFNγ released in the presence of cells pulsed with hTERT865–873 peptide after subtraction of values obtained in the presence of control peptide-pulsed cells. Spearman rank correlation between dextramer positivity and hIFNγ release for “TERT responders” patients (inset). C, Kaplan–Meier analysis of “TERT responders” and “TERT nonresponders” patients analyzed in relation to the time (months) to first treatment. D, summary of mouse hTERT865–873–specific TCR sequence used to engineer human T cells. E, flow-cytometric analysis of mouse Vβ3 chain expression and dextramer positivity of hTERT865–873– and HCV1406–1415–specific, TCR-engineered T cells. F, functional avidity evaluation of the endogenous, HLA-A2–restricted hTERT-specific T cells isolated from two representative “TERT responders” patients (blue lines) and the hTERT865–873–specific, TCR-engineered T cells (black line) after coculture in the presence of T2 cells loaded with varying hTERT865–873 peptide concentrations. Values were normalized to IFNγ released by the “TERT responder” patient with highest response. Red dashed line, the 50% of maximum response. G, analysis of fratricidal activity of hTERT865–873–specific, TCR-engineered T cells against either PBMCs or HCV1406–1415–TCR-transduced T cells pulsed with hTERT865–873 peptide, hCMV495–503 peptide, or left unpulsed. Data are mean ± SD of 1 of 3 independent experiments. ANOVA test.

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We therefore explored the feasibility to redirect human T lymphocytes by genetic engineering based on de novo expression of a high-avidity, HLA-A2–restricted, hTERT-specific TCR. Briefly, we first isolated a mouse CTL clone expressing a TCR for human HLA-A2–restricted hTERT865–873 epitope from a polyclonal, in vitro stabilized T cell population obtained from HLA-A2 transgenic mice vaccinated toward hTERT (17). The sequences of the α and β chains of the TCR (Fig. 3D) were then cloned by Vα- and Vβ-specific primer panels, amplified by PCR and the products of the reaction were sequenced and inserted into a retroviral vector (34) able to transduce naïve T cells. After expansion of human T cells in vitro transduced with either anti-hTERT or anti-hHCV (as control) TCR-encoding retroviruses (36), we checked the expression of the mouse TCR Vβ chain (mVβ3), a component of the hTERT865–873–specific TCR, on T-cell surface. (Fig. 3E). We further confirmed the presence of the correct, antigen-specific TCR on transduced T cells by dextramer staining. (Fig. 3E). The engineered hTERT865–873–specific T cells displayed a much higher avidity TCR compared with the endogenous TCRs expressed by T lymphocytes isolated from B-CLL patients, confirming the expected, intrinsic low avidity of the endogenous T-cell repertoire for self-antigens (Fig. 3F). Finally, we verified that hTERT865–873–specific TCR-engineered T cells did not cause a catastrophic MHC-restricted T-cell fratricide, as recently shown for survivin-specific, TCR-engineered T lymphocytes (37). The hTERT865–873–specific, TCR-engineered T cells recognized HLA-A2+ PBMCs and activated T-lymphocytes (infected with retrovirally encoded anti-hHCV TCR) only when the target T cells were pulsed with the hTERT865–873 peptide; no recognition of PBMCs and activated T lymphocytes, either unpulsed or pulsed with the control hHCV1406–1415 peptide, could be detected (Fig. 3G). These data indicate that hTERT865–873-specific, TCR-engineered T lymphocytes cannot kill human proliferating T lymphocytes.

Engineered hTERT865–873–specific, TCR-engineered T cells efficiently restrain human solid tumor and B-CLL in vivo growth

Both CD4+ and CD8+ T lymphocytes engineered with hTERT865–873–specific TCR showed an effector memory phenotype (CD45RACD62LCCR7; Supplementary Fig. S4A) characterized by high expression of CD69/CD44/CD38/CD25 and HLA-DR markers and low expression of exhaustion markers (LAG3/PD-1/Tim-3) compared with the specific isotype controls (Supplementary Fig. S4C). Moreover, CD4+ T cells were polarized toward IFNγ-producing Th1 cells (Supplementary Fig. S4D). We selected several tumor cell lines of different histology, comprising hematologic malignancies such as myeloma (U266), B-CLL (JVM13 and MEC-1), and Burkitt lymphoma cells (DG-75), and solid tumors, such as melanoma (SK23MEL), breast carcinoma (MDA-MB-231), and colon carcinoma (SW480) cells. The selected tumor cell lines showed high levels of TERT activity and heterogeneous levels of both TERT protein and HLA-A2 membrane complex (Fig. 4A). The hTERT865–873–specific, TCR-engineered T cells were capable of recognizing in vitro HLA-A2+ B-cell tumor cell lines with the expected exception of HLA-A2 MEC-1 (Fig 4B). The broad antitumor therapeutic activity of hTERT865–873–specific, TCR-engineered T cells was confirmed in vivo in immunodeficient NOG mice engrafted s.c. with five different HLA-A2+ tumors. Both adoptively transferred hTERT865–873– and hHCV1406–1415–specific, TCR-transduced T cells comparably migrated to the tumor site (Fig. 4C), although only hTERT-specific ACT significantly controlled tumor growth and prolonged mice survival (Fig 4D and Supplementary Fig. S4E). These data clearly advocate the potential effectiveness of TERT-based ACT approach to treat different human cancers.

Figure 4.

Therapeutic efficacy of engineered hTERT865–873–specific, TCR-engineered T cells toward HLA-A2+ hematologic and solid transplantable tumors. A, characterization of TERT and HLA expression in malignant cells of different histotypes. TERT protein expression was evaluated by Western blot. TERT activity was measured by the TRAP assay, and HLA-A2 expression levels were evaluated by flow cytometry (numbers indicate mean fluorescence index). B, cell killing activity of hTERT865-873–specific, TCR-engineered T cells was evaluated by the 51Cr release assay (left) and hIFNγ ELISA (right). Data are mean ± SD of three independent experiments. C, flow cytometric evaluation of tumor-infiltrating infused engineered T cells. Data are mean ± SD from 3 mice per group. D, therapeutic impact of hTERT865–873–specific, TCR-engineered T cell on tumor growth (top) promoting tumor-bearing mouse survival (bottom). Data are mean ± SD from 1 of 3 experiments. SK23mel, hTERT ACT (n = 8) versus hHCV ACT (n = 4); SW480, hTERT ACT (n = 6) versus hHCV ACT (n = 6); MDA-MB-231, hTERT ACT (n = 7) versus hHCV ACT (n = 7).

Figure 4.

Therapeutic efficacy of engineered hTERT865–873–specific, TCR-engineered T cells toward HLA-A2+ hematologic and solid transplantable tumors. A, characterization of TERT and HLA expression in malignant cells of different histotypes. TERT protein expression was evaluated by Western blot. TERT activity was measured by the TRAP assay, and HLA-A2 expression levels were evaluated by flow cytometry (numbers indicate mean fluorescence index). B, cell killing activity of hTERT865-873–specific, TCR-engineered T cells was evaluated by the 51Cr release assay (left) and hIFNγ ELISA (right). Data are mean ± SD of three independent experiments. C, flow cytometric evaluation of tumor-infiltrating infused engineered T cells. Data are mean ± SD from 3 mice per group. D, therapeutic impact of hTERT865–873–specific, TCR-engineered T cell on tumor growth (top) promoting tumor-bearing mouse survival (bottom). Data are mean ± SD from 1 of 3 experiments. SK23mel, hTERT ACT (n = 8) versus hHCV ACT (n = 4); SW480, hTERT ACT (n = 6) versus hHCV ACT (n = 6); MDA-MB-231, hTERT ACT (n = 7) versus hHCV ACT (n = 7).

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We treated leukemic-bearing mice, systemically engrafted with JVM13-Luc cells, for 3 consecutive weeks with either hTERT865–873– or hHCV1406–1415–specific, TCR-transduced T cells, weekly monitoring tumor cell expansion by bioluminescence imaging. The hTERT-specific ACT induced a significant decrease in radiance signals in leukemia-bearing mice compared with mice receiving hHCV-specific ACT (Fig. 5A). Thirty-five days after the first treatment, we assessed tumor spreading to different organs (Fig. 5B–C). Moreover, we performed IHC of human CD20+ cells in the spleen of ACT-treated mice (Fig. 5D). All these analyses showed that engineered hTERT865–873–specific, TCR-engineered T cells induced a significant reduction in human B-CLL accumulation.

Figure 5.

hTERT865–873–specific, TCR-engineered T cells restrain human B-CLL progression. A, therapeutic efficacy of hTERT-specific ACT in controlling JVM-13 Luc+ cell expansion (n = 5 each; hTERT vs. hHCV ACT, P = 0.031; RM ANOVA test). B, bioluminescence imaging of tumor cells into different organs of tumor-free NOG mice (black), hTERT-specific ACT-treated mice (blue), and hHCV-specific ACT-treated mice (red). C, flow cytometric evaluation of infiltrating hCD19+ cells in different organs (BM, P < 0.001; spleen, P < 0.001; lung, P = 0.044; liver, P < 0.039). D, IHC analysis of hCD20 expression in the spleen of both hTERT- and hHCV-specific ACT-treated mice. Bars, 50 μm. All data are mean ± SD of a representative experiment of two independent experiments. Student t test.

Figure 5.

hTERT865–873–specific, TCR-engineered T cells restrain human B-CLL progression. A, therapeutic efficacy of hTERT-specific ACT in controlling JVM-13 Luc+ cell expansion (n = 5 each; hTERT vs. hHCV ACT, P = 0.031; RM ANOVA test). B, bioluminescence imaging of tumor cells into different organs of tumor-free NOG mice (black), hTERT-specific ACT-treated mice (blue), and hHCV-specific ACT-treated mice (red). C, flow cytometric evaluation of infiltrating hCD19+ cells in different organs (BM, P < 0.001; spleen, P < 0.001; lung, P = 0.044; liver, P < 0.039). D, IHC analysis of hCD20 expression in the spleen of both hTERT- and hHCV-specific ACT-treated mice. Bars, 50 μm. All data are mean ± SD of a representative experiment of two independent experiments. Student t test.

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To further explore the usefulness of this immunotherapeutic approach from a translational standpoint, we evaluated the hTERT presence in PBMCs isolated from B-CLL patients and age-matched HDs. PBMCs of B-CLL patients exhibited higher hTERT expression and enzymatic activity levels compared with HD PBMCs (Supplementary Fig. S5A). Moreover, B cells isolated from HLA-A2+ B-CLL patients were also recognized in vitro by hTERT865–873–specific, TCR-engineered T lymphocytes similarly to the JVM13 cell line, while HLA-A2 B-CLL PBMCs as well as HLA-A2+ HD PBMCs were not recognized (Fig. 6A). We finally tested the anti-leukemic activity of ACT against B-CLL patients' PBMCs in vivo. To this aim, immunodeficient NOG mice, previously humanized with allogeneic BM-derived CD34+ cells, were engrafted with either HLA-A2+ or HLA-A2 PBMCs from B-CLL patients previously classified as TERT responders (Fig. 3C) and treated with three weekly courses of ACT (Supplementary Fig. S5B). In mice inoculated with HLA-A2+ PBMCs from B-CLL patients, the total number of circulating hTERT865–873–specific, TCR-engineered T cells inversely correlated with the percentage of CD19+HLA-A2+ malignant B cells (Fig. 6B, left). On the contrary, ACT with hTERT865–873–specific, TCR-engineered T cells had no significant effects on the number of hCD19+ cells developed in mice engrafted with HLA-A2 B-CLL (Fig. 6B, right). One month after B-CLL engraftment, we evaluated malignant B-cell infiltration in both spleen and BM. The hTERT-specific ACT in HLA-A2+ B-CLL–bearing mice visibly controlled the leukemia progression (Fig. 6C). Conversely, hTERT-specific ACT did not control the neoplastic progression in HLA-A2 B-CLL–bearing mice (Fig. 6D), although we detected the same amount of human CD8+ T-cell infiltration, as measured by flow cytometry (data not shown).

Figure 6.

hTERT865–873–specific, TCR-engineered T cells selectively recognize HLA-A2+ patients' B-CLL cells. A, B-CLL patient isolated B cells were recognized in vitro by hTERT865–873–specific, TCR-engineered as assayed both by flow-cytometry cytotoxicity assay (top) and hIFNγ release assay (bottom). Data are mean ± SD of three independent experiments: hTERT865–873–pulsed HLA-A2+ HD B cells (n = 20; CTRL+); hHCV1406–1415–pulsed HLA-A2+ HD B cells (n = 20; CTRL); HLA-A2+ HD B cells (n = 20); HLA-A2 B cells from B-CLL patients (n = 10); HLA-A2+ B cells from B-CLL patients (n = 10). ANOVA test. B, humanized NOG mice, challenged with PBMCs isolated from B-CLL patients (HLA-A2+ patient, left; HLA-A2 patient, right), were treated with hTERT- or hHCV-specific ACTs. Spearman rank correlation between circulating malignant B-cells and circulating infused engineered T cells (hTERT ACT, n = 9; hHCV ACT, n = 7; circles, evaluation after the second ACT; triangles, evaluation after the third ACT). C, IHC (left) and flow cytometric (right) evaluation of HLA-A2+ leukemic cells after hTERT- or hHCV-specific ACT. Bars, 100 μm; Student t test. D, as indicated in C, evaluation of HLA-A2 leukemic cells after hTERT- or hHCV-specific ACT. Bars, 100 μm. Student t test.

Figure 6.

hTERT865–873–specific, TCR-engineered T cells selectively recognize HLA-A2+ patients' B-CLL cells. A, B-CLL patient isolated B cells were recognized in vitro by hTERT865–873–specific, TCR-engineered as assayed both by flow-cytometry cytotoxicity assay (top) and hIFNγ release assay (bottom). Data are mean ± SD of three independent experiments: hTERT865–873–pulsed HLA-A2+ HD B cells (n = 20; CTRL+); hHCV1406–1415–pulsed HLA-A2+ HD B cells (n = 20; CTRL); HLA-A2+ HD B cells (n = 20); HLA-A2 B cells from B-CLL patients (n = 10); HLA-A2+ B cells from B-CLL patients (n = 10). ANOVA test. B, humanized NOG mice, challenged with PBMCs isolated from B-CLL patients (HLA-A2+ patient, left; HLA-A2 patient, right), were treated with hTERT- or hHCV-specific ACTs. Spearman rank correlation between circulating malignant B-cells and circulating infused engineered T cells (hTERT ACT, n = 9; hHCV ACT, n = 7; circles, evaluation after the second ACT; triangles, evaluation after the third ACT). C, IHC (left) and flow cytometric (right) evaluation of HLA-A2+ leukemic cells after hTERT- or hHCV-specific ACT. Bars, 100 μm; Student t test. D, as indicated in C, evaluation of HLA-A2 leukemic cells after hTERT- or hHCV-specific ACT. Bars, 100 μm. Student t test.

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Adoptive transfer of hTERT865–873–specific, TCR-engineered T cells induces the depletion of mature granulocytes but does not affect hematopoietic stem cells

Even though engineered T cell–based therapies have shown long-term efficacy and promising curative potential for the treatment of cancer, several “on-target, off-tumor” toxicities have been reported (38). We therefore investigated the toxicity of hTERT-based immunotherapeutic approach toward the hematopoietic compartment. Although human CD34+HLA-A2+ cells were dimly recognized by hTERT865–873–specific, TCR-engineered T cells upon in vitro coculture (Fig. 7A), they maintained their in vitro ability to proliferate and differentiate into colonies in a semisolid medium (Fig. 7B) and preserved their multipotency once injected into γ-irradiated, immunodeficient NOG mice (Fig. 7C). Moreover, we treated human immune reconstituted (HIR) mice, obtained by transplantation of human HLA-A2+ CD34+ cells into immunodeficient NOG mice, with two consecutive ACTs (Supplementary Fig. S5C). Seventeen weeks after CD34+ cell engraftment, the total number of human CD45+ cells in the spleen was not affected by hTERT-specific ACT (Fig. 7D–E), while they were significantly reduced in the BM compared with mice receiving control ACT (Fig. 7E). The relative distribution among human leukocytes was maintained in the spleen (Fig. 7F, left), as well as the percentage of both human granulocytes and monocytes among myeloid cells (CD45+CD33+SSChigh and CD45+CD33+SSClow, respectively; Fig. 7F, right). Moreover, we could not identify any alteration of relative distribution of myeloid and lymphoid cells in BM (Fig. 7G, left), except for a significant contraction in human granulocytes (Fig. 7G, right). Indeed, among granulocytic cell subsets, we observed a nearly complete deletion of the more mature CD45+CD11b+CD16+ human myeloid cells in mice treated with hTERT-specific ACT (Fig. 7H). To confirm a potential hTERT-specific, ACT-linked toxicity on myeloid mature cell subsets, we purified from human BM aspirate of HDs the three main granulocytic maturation cell fractions (myeloblasts, promyelocytes, and granulocytes; ref. 39). Only the more mature cell population, i.e., CD45+CD11b+CD16+, was significantly recognized in vitro by hTERT865–873–specific, TCR-engineered T cells (Fig. 7I), although no difference in telomerase enzyme activity levels was detected among the different cell fractions (data not shown). Thus, hTERT865–873–specific, TCR-engineered T cells ignore progenitors and specifically target only mature myeloid subsets, potentially limiting the induction of a prolonged adverse neutropenia, a common consequence of conventional chemotherapeutic treatments.

Figure 7.

hTERT865–873–specific, TCR-engineered T cells do not reduce stem cell multipotency but induce a selective depletion of mature BM granulocytic populations. A, HLA-A2+hCD34+ cells and either hTERT865–873– (CTRL+) or hHCV1406–1415–peptide loaded (CTRL) T2 cells were preincubated in vitro with hTERT865–873– or control HCV1406–1415–TCR-engineered T cells. Levels of released hIFNγ were evaluated by ELISA. Data are mean ± SD of three independent experiments. ANOVA test. B, after incubation, isolated hCD34+ cells were used in a colony-forming unit (CFU) assay. Data are mean ± SD of three independent experiments. Student t test. C, evaluation of circulating hCD45+ cells in NOG, engrafted with hCD34+ cells preincubated in vitro with hTERT865-873- or HCV1406-1415-specific, TCR-engineered T cells. Data are from 1 of 2 independent experiments (n = 6). Statistical analysis was performed with RM ANOVA: CD34+/hTERT ACT vs. CD34+/hHCV ACT, P = 0.32. D, distribution of human CD45+ leukocytes in the spleen of humanized NOG mice treated with hTERT865–873– (n = 7) or control HCV1406–1415– (n = 6) specific, TCR-engineered T cells. Bars, 50 μm. Data are mean ± SD of 1 of 2 independent experiments. Student t test, P = 0.64. E, flow cytometric analysis of hCD45+ splenocytes and BM cells isolated from ACT-treated HIR mice. F, relative proportions of human splenic leukocytic populations (left) and percentages of CD33+/SSChigh granulocytes and CD33+/SSClow monocytes (right). G, relative proportions of human cell populations in BM (left), with analysis of myeloid subpopulations (right). H, relative proportion of human BM granulocytes divided according to their maturation stages with CD11b and CD16 markers. Student t test. I, human BM cells derived from healthy donors were divided by FACS sorting in three main populations: CD11bCD16 (orange), CD11b+CD16 (green), and CD11b+CD16+ (pink). These three cell subsets were incubated in vitro in the presence of either hTERT865–873– or control HCV1406–1415–specific, TCR-engineered T cells. IFNγ levels were assessed by ELISA. Bars, 50 μm. Data are mean ± SD of four experiments. ANOVA test.

Figure 7.

hTERT865–873–specific, TCR-engineered T cells do not reduce stem cell multipotency but induce a selective depletion of mature BM granulocytic populations. A, HLA-A2+hCD34+ cells and either hTERT865–873– (CTRL+) or hHCV1406–1415–peptide loaded (CTRL) T2 cells were preincubated in vitro with hTERT865–873– or control HCV1406–1415–TCR-engineered T cells. Levels of released hIFNγ were evaluated by ELISA. Data are mean ± SD of three independent experiments. ANOVA test. B, after incubation, isolated hCD34+ cells were used in a colony-forming unit (CFU) assay. Data are mean ± SD of three independent experiments. Student t test. C, evaluation of circulating hCD45+ cells in NOG, engrafted with hCD34+ cells preincubated in vitro with hTERT865-873- or HCV1406-1415-specific, TCR-engineered T cells. Data are from 1 of 2 independent experiments (n = 6). Statistical analysis was performed with RM ANOVA: CD34+/hTERT ACT vs. CD34+/hHCV ACT, P = 0.32. D, distribution of human CD45+ leukocytes in the spleen of humanized NOG mice treated with hTERT865–873– (n = 7) or control HCV1406–1415– (n = 6) specific, TCR-engineered T cells. Bars, 50 μm. Data are mean ± SD of 1 of 2 independent experiments. Student t test, P = 0.64. E, flow cytometric analysis of hCD45+ splenocytes and BM cells isolated from ACT-treated HIR mice. F, relative proportions of human splenic leukocytic populations (left) and percentages of CD33+/SSChigh granulocytes and CD33+/SSClow monocytes (right). G, relative proportions of human cell populations in BM (left), with analysis of myeloid subpopulations (right). H, relative proportion of human BM granulocytes divided according to their maturation stages with CD11b and CD16 markers. Student t test. I, human BM cells derived from healthy donors were divided by FACS sorting in three main populations: CD11bCD16 (orange), CD11b+CD16 (green), and CD11b+CD16+ (pink). These three cell subsets were incubated in vitro in the presence of either hTERT865–873– or control HCV1406–1415–specific, TCR-engineered T cells. IFNγ levels were assessed by ELISA. Bars, 50 μm. Data are mean ± SD of four experiments. ANOVA test.

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We show here the strength and safety of a broadly applicable immunotherapy protocol based on engineered T cells generated by transduction with a high-affinity TCR capable of recognizing the complex formed by hTERT865–873 peptide and HLA-A2 molecule. Telomerase can be regarded as a universal TAA: it contributes to sustain tumor cell survival (40) and prevent apoptosis elicited by antiproliferative agents (41). Furthermore, hTERT is immunogenic (27) and, indeed, five HLA-A2–restricted epitopes have already been identified: I540 (19), R865 (20), 572Ya and 988Ya (42), and R38a (22). The first identified peptide, I540, was also tested in different cancer vaccination clinical trials that showed a modest impact on tumor control (26, 27). To the best of our knowledge, we show here for the first time that a specific cohort of B-CLL patients, with a trend toward a less aggressive leukemia, presented a specific, endogenous response toward the R865 epitope that was not detectable in HDs (Fig. 3A–B). Our data diverge from results published about I540 epitope, which is able to promote a comparable immune response in both tumor patients and normal donors (43). The differences between I540 and R865 epitopes might be related to a different baseline of immune tolerance between the two epitopes. Moreover, the TCR affinity of the endogenous anti-hTERT865–872 T cells isolated from B-CLL patients is deeply low (Fig. 3F), which makes it unrealistic to isolate high-avidity T cells from patients to develop an ACT protocol. So far, only one study reported the redirected T cell–based adoptive immunotherapy targeting hTERT with a TCR isolated from human CTLs (21), generated from PBMCs of HDs in vitro stimulated with HLA-A24:02–restricted nonameric hTERT461–469 peptide (44). However, the overall functional avidity to hTERT461–469 peptide evaluated for transduced T cells was about 10−7 mol/L, which is modest compared with the TCR affinity of our engineered T cells, which displayed 50% of their maximum response at 10−14 mol/L peptide concentration (Fig. 3F). Transduced anti-hTERT T cells specifically target tumor cells, such as leukemic B cells both in vitro and in vivo, without affecting normal B-cell development. Indeed, anti-TERT T cells do not alter dramatically stem cell differentiation, suggesting a lower toxicity compared with common chemotherapies that normally promote severe myeloid precursor depletion in treated patients, often requiring the administration of support therapy to restore normal hematopoiesis. The ability of TERT targeting to control B-CLL progression is also confirmed by our data using IgH.TEμ mice; in fact, polyclonal anti-TERT CTLs transfer was able to significantly improve the leukemia-bearing mouse survival (Fig 2I). The ability of hTERT865–873–specific, TCR-engineered T cells to eliminate mature granulocytes hints at an extension of this therapy toward human acute myeloid leukemia (AML), that is characterized by a strong TERT activity directly correlating with poor outcome of the disease (45). Another potential target for hTERT-specific ACT is represented by B-cell acute lymphoblastic leukemia, in which TERT locus is recurrently targeted by somatic chromosomal translocations (46) and TERT expression is a marker associated with inferior clinical outcome (47). Finally, our data about the therapeutic ability of hTERT-specific ACT to efficiently control cancer progression of different solid tumors (Fig. 4B) suggest how the transduced anti-hTERT T cells could be a potential “off-the-shelf” reagent applicable to treat many oncologic diseases.

TERT is physiologically activated in a limited number of human normal cell populations (48). Unfortunately, experimental models that can predict potential toxic effects against these human cells are currently not available and some off-target activity can be completely unpredictable (49). However, to control off-target activity and mitigate excessive in vivo T-cell activation/expansion after systemic infusion, which might induce a lethal cytokine storm (50), we plan to develop an antidote based on the administration of antibodies specific for the mouse Vβ3 chain of the engineered TCR. Moreover, to limit direct toxicity, uncontrolled growth and malignant transformation of hTERT865–873–specific, TCR-engineered T cells, we plan to insert a suicide gene, such as the inducible caspase-9 gene (iCasp9), which can be triggered in the case of unfavorable events (51). The clinic impact of these new strategies was recently demonstrated to control the graft versus host disease (GVHD) symptoms in acute leukemia relapsed patients, after allogeneic stem-cell transplant (52). Importantly, we also excluded a fratricide effect of engineered T cells, as well as the elimination of activated endogenous T cells, which might limit the power of immunotherapy. Finally, for a clinical point of view, it could be feasible to isolate a fully humanized TCR against hTERT by immunization of antigen-negative humanized mice that can generate optimal affinity TCRs for T-cell therapy (16).

No potential conflicts of interest were disclosed.

Conception and design: S. Sandri, C. Cavallini, M.T. Scupoli, S. Sartoris, V. Bronte, S. Ugel

Development of methodology: S. Sandri, G. Fracasso, R.W. Hendriks, S. Ugel

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Sandri, F. De Sanctis, F. Boschi, G. Ferrarini, C. Cavallini, M.T. Scupoli, M.I. Nishimura

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Sandri, A. Lamolinara, F. De Sanctis, F. Boschi, C. Cavallini, M.T. Scupoli, M. Iezzi, M.I. Nishimura, V. Bronte, S. Ugel

Writing, review, and/or revision of the manuscript: S. Sandri, M.I. Nishimura, V. Bronte, S. Ugel

Study supervision: S. Sartoris, V. Bronte

Other (performed research and provided essential new reagents): S. Bobisse, K. Moxley

Other (assisted with experimental design of in vivo imaging study): A. Sbarbati

Other (analyzed tissue sections by immunohistochemistry and interpreted the data): A. Lamolinara

The authors thank Elisa Zoratti, Mauro Giacca, Lorena Zentilin, Martina Tinelli, Loredana Ruggeri, Ornella Poffe, Rosalinda Trovato, Alessandra Fiore, and Cristina Anselmi for technical help.

This work was supported by grants from the Italian Ministry of Health, Italian Ministry of Education, Universities, and Research (FIRB cup: B31J11000420001), Italian Association for Cancer Research (AIRC; grants 6599, 12182, and 14103), National Cancer Institute (P01 CA154778 to M.I. Nishimura), and Dutch Cancer Society (R.W. Hendriks).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
June
CH
,
Riddell
SR
,
Schumacher
TN
. 
Adoptive cellular therapy: a race to the finish line
.
Sci Transl Med
2015
;
7
:
280ps7
.
2.
Rosenberg
SA
,
Restifo
NP
. 
Adoptive cell transfer as personalized immunotherapy for human cancer
.
Science
2015
;
348
:
62
8
.
3.
Dudley
ME
,
Wunderlich
JR
,
Robbins
PF
,
Yang
JC
,
Hwu
P
,
Schwartzentruber
DJ
, et al
Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes
.
Science
2002
;
298
:
850
4
.
4.
Rosenberg
SA
,
Yang
JC
,
Sherry
RM
,
Kammula
US
,
Hughes
MS
,
Phan
GQ
, et al
Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy
.
Clin Cancer Res
2011
;
17
:
4550
7
.
5.
Rosenberg
SA.
Cell transfer immunotherapy for metastatic solid cancer–what clinicians need to know
.
Nat Rev Clin Oncol
2011
;
8
:
577
85
.
6.
Robbins
PF
,
Lu
YC
,
El-Gamil
M
,
Li
YF
,
Gross
C
,
Gartner
J
, et al
Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells
.
Nat Med
2013
;
19
:
747
52
.
7.
Robbins
PF
,
Morgan
RA
,
Feldman
SA
,
Yang
JC
,
Sherry
RM
,
Dudley
ME
, et al
Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1
.
J Clin Oncol
2011
;
29
:
917
24
.
8.
Grupp
SA
,
Kalos
M
,
Barrett
D
,
Aplenc
R
,
Porter
DL
,
Rheingold
SR
, et al
Chimeric antigen receptor-modified T cells for acute lymphoid leukemia
.
N Engl J Med
2013
;
368
:
1509
18
.
9.
Roszkowski
JJ
,
Lyons
GE
,
Kast
WM
,
Yee
C
,
Van Besien
K
,
Nishimura
MI
. 
Simultaneous generation of CD8+ and CD4+ melanoma-reactive T cells by retroviral-mediated transfer of a single T-cell receptor
.
Cancer Res
2005
;
65
(4):
1570
6
.
10.
Johnson
LA
,
Heemskerk
B
,
Powell
DJ
 Jr.
,
Cohen
CJ
,
Morgan
RA
,
Dudley
ME
, et al
Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes
.
J Immunol
2006
;
177
:
6548
59
.
11.
Theobald
M
,
Biggs
J
,
Hernandez
J
,
Lustgarten
J
,
Labadie
C
,
Sherman
LA
. 
Tolerance to p53 by A2.1-restricted cytotoxic T lymphocytes
.
J Exp Med
1997
;
185
:
833
41
.
12.
Linnemann
C
,
van Buuren
MM
,
Bies
L
,
Verdegaal
EM
,
Schotte
R
,
Calis
JJ
, et al
High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma
.
Nat Med
2015
;
21
:
81
5
.
13.
Wilde
S
,
Sommermeyer
D
,
Frankenberger
B
,
Schiemann
M
,
Milosevic
S
,
Spranger
S
, et al
Dendritic cells pulsed with RNA encoding allogeneic MHC and antigen induce T cells with superior antitumor activity and higher TCR functional avidity
.
Blood
2009
;
114
:
2131
9
.
14.
Carmon
L
,
Bobilev-Priel
I
,
Brenner
B
,
Bobilev
D
,
Paz
A
,
Bar-Haim
E
, et al
Characterization of novel breast carcinoma-associated BA46-derived peptides in HLA-A2.1/D(b)-beta2m transgenic mice
.
J Clin Invest
2002
;
110
:
453
62
.
15.
Kuball
J
,
Schmitz
FW
,
Voss
RH
,
Ferreira
EA
,
Engel
R
,
Guillaume
P
, et al
Cooperation of human tumor-reactive CD4+ and CD8+ T cells after redirection of their specificity by a high-affinity p53A2.1-specific TCR
.
Immunity
2005
;
22
:
117
29
.
16.
Obenaus
M
,
Leitao
C
,
Leisegang
M
,
Chen
X
,
Gavvovidis
I
,
van der Bruggen
P
, et al
Identification of human T-cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice
.
Nat Biotechnol
2015
;
33
:
402
7
.
17.
Ugel
S
,
Scarselli
E
,
Iezzi
M
,
Mennuni
C
,
Pannellini
T
,
Calvaruso
F
, et al
Autoimmune B-cell lymphopenia after successful adoptive therapy with telomerase-specific T lymphocytes
.
Blood
2010
;
115
:
1374
84
.
18.
Hiyama
E
,
Hiyama
K
. 
Telomerase as tumor marker
.
Cancer Lett
2003
;
194
:
221
33
.
19.
Vonderheide
RH
,
Hahn
WC
,
Schultze
JL
,
Nadler
LM
. 
The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes
.
Immunity
1999
;
10
:
673
9
.
20.
Minev
B
,
Hipp
J
,
Firat
H
,
Schmidt
JD
,
Langlade-Demoyen
P
,
Zanetti
M
. 
Cytotoxic T cell immunity against telomerase reverse transcriptase in humans
.
Proc Natl Acad Sci U S A
2000
;
97
:
4796
801
.
21.
Arai
J
,
Yasukawa
M
,
Ohminami
H
,
Kakimoto
M
,
Hasegawa
A
,
Fujita
S
. 
Identification of human telomerase reverse transcriptase-derived peptides that induce HLA-A24-restricted antileukemia cytotoxic T lymphocytes
.
Blood
2001
;
97
:
2903
7
.
22.
Thorn
M
,
Wang
M
,
Kloverpris
H
,
Schmidt
EG
,
Fomsgaard
A
,
Wenandy
L
, et al
Identification of a new hTERT-derived HLA-A*0201 restricted, naturally processed CTL epitope
.
Cancer Immunol, Immunother: CII
2007
;
56
:
1755
63
.
23.
Cheever
MA
,
Allison
JP
,
Ferris
AS
,
Finn
OJ
,
Hastings
BM
,
Hecht
TT
, et al
The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational research
.
Clin Cancer Res
2009
;
15
(17):
5323
37
.
24.
Brunsvig
PF
,
Aamdal
S
,
Gjertsen
MK
,
Kvalheim
G
,
Markowski-Grimsrud
CJ
,
Sve
I
, et al
Telomerase peptide vaccination: a phase I/II study in patients with non-small cell lung cancer
.
Cancer Immunol, Immunother: CII
2006
;
55
:
1553
64
.
25.
Bernhardt
SL
,
Gjertsen
MK
,
Trachsel
S
,
Moller
M
,
Eriksen
JA
,
Meo
M
, et al
Telomerase peptide vaccination of patients with non-resectable pancreatic cancer: A dose escalating phase I/II study
.
Br J Cancer
2006
;
95
:
1474
82
.
26.
Vonderheide
RH
,
Domchek
SM
,
Schultze
JL
,
George
DJ
,
Hoar
KM
,
Chen
DY
, et al
Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes
.
Clin Cancer Res
2004
;
10
:
828
39
.
27.
Domchek
SM
,
Recio
A
,
Mick
R
,
Clark
CE
,
Carpenter
EL
,
Fox
KR
, et al
Telomerase-specific T-cell immunity in breast cancer: effect of vaccination on tumor immunosurveillance
.
Cancer Res
2007
;
67
:
10546
55
.
28.
Nair
SK
,
Heiser
A
,
Boczkowski
D
,
Majumdar
A
,
Naoe
M
,
Lebkowski
JS
, et al
Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells
.
Nat Med
2000
;
6
:
1011
7
.
29.
Su
Z
,
Dannull
J
,
Yang
BK
,
Dahm
P
,
Coleman
D
,
Yancey
D
, et al
Telomerase mRNA-transfected dendritic cells stimulate antigen-specific CD8+ and CD4+ T cell responses in patients with metastatic prostate cancer
.
J Immunol
2005
;
174
:
3798
807
.
30.
Damle
RN
,
Batliwalla
FM
,
Ghiotto
F
,
Valetto
A
,
Albesiano
E
,
Sison
C
, et al
Telomere length and telomerase activity delineate distinctive replicative features of the B-CLL subgroups defined by immunoglobulin V gene mutations
.
Blood
2004
;
103
:
375
82
.
31.
Terrin
L
,
Trentin
L
,
Degan
M
,
Corradini
I
,
Bertorelle
R
,
Carli
P
, et al
Telomerase expression in B-cell chronic lymphocytic leukemia predicts survival and delineates subgroups of patients with the same igVH mutation status and different outcome
.
Leukemia
2007
;
21
:
965
72
.
32.
ter Brugge
PJ
,
Ta
VB
,
de Bruijn
MJ
,
Keijzers
G
,
Maas
A
,
van Gent
DC
, et al
A mouse model for chronic lymphocytic leukemia based on expression of the SV40 large T antigen
.
Blood
2009
;
114
:
119
27
.
33.
Hallek
M
,
Cheson
BD
,
Catovsky
D
,
Caligaris-Cappio
F
,
Dighiero
G
,
Dohner
H
, et al
Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines
.
Blood
2008
;
111
:
5446
56
.
34.
Norell
H
,
Zhang
Y
,
McCracken
J
,
Martins da Palma
T
,
Lesher
A
,
Liu
Y
, et al
CD34-based enrichment of genetically engineered human T cells for clinical use results in dramatically enhanced tumor targeting
.
Cancer Immunol, Immunother: CII
2010
;
59
:
851
62
.
35.
Bagnara
D
,
Kaufman
MS
,
Calissano
C
,
Marsilio
S
,
Patten
PE
,
Simone
R
, et al
A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease
.
Blood
2011
;
117
:
5463
72
.
36.
Zhang
Y
,
Liu
Y
,
Moxley
KM
,
Golden-Mason
L
,
Hughes
MG
,
Liu
T
, et al
Transduction of human T cells with a novel T-cell receptor confers anti-HCV reactivity
.
PLoS Pathogens
2010
;
6
:
e1001018
.
37.
Leisegang
M
,
Wilde
S
,
Spranger
S
,
Milosevic
S
,
Frankenberger
B
,
Uckert
W
, et al
MHC-restricted fratricide of human lymphocytes expressing survivin-specific transgenic T cell receptors
.
J Clin Invest
2010
;
120
:
3869
77
.
38.
Tey
SK
. 
Adoptive T-cell therapy: adverse events and safety switches
.
Clin Transl Immunol
2014
;
3
:
e17
.
39.
Solito
S
,
Falisi
E
,
Diaz-Montero
CM
,
Doni
A
,
Pinton
L
,
Rosato
A
, et al
A human promyelocytic-like population is responsible for the immune suppression mediated by myeloid-derived suppressor cells
.
Blood
2011
;
118
:
2254
65
.
40.
Cao
Y
,
Li
H
,
Deb
S
,
Liu
JP
. 
TERT regulates cell survival independent of telomerase enzymatic activity
.
Oncogene
2002
;
21
:
3130
8
.
41.
Rahaman
SO
,
Harbor
PC
,
Chernova
O
,
Barnett
GH
,
Vogelbaum
MA
,
Haque
SJ
. 
Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells
.
Oncogene
2002
;
21
:
8404
13
.
42.
Scardino
A
,
Gross
DA
,
Alves
P
,
Schultze
JL
,
Graff-Dubois
S
,
Faure
O
, et al
HER-2/neu and hTERT cryptic epitopes as novel targets for broad spectrum tumor immunotherapy
.
J Immunol
2002
;
168
:
5900
6
.
43.
Vonderheide
RH
,
Schultze
JL
,
Anderson
KS
,
Maecker
B
,
Butler
MO
,
Xia
Z
, et al
Equivalent induction of telomerase-specific cytotoxic T lymphocytes from tumor-bearing patients and healthy individuals
.
Cancer Res
2001
;
61
:
8366
70
.
44.
Miyazaki
Y
,
Fujiwara
H
,
Asai
H
,
Ochi
F
,
Ochi
T
,
Azuma
T
, et al
Development of a novel redirected T-cell-based adoptive immunotherapy targeting human telomerase reverse transcriptase for adult T-cell leukemia
.
Blood
2013
;
121
:
4894
901
.
45.
Aalbers
AM
,
Calado
RT
,
Young
NS
,
Zwaan
CM
,
Wu
C
,
Kajigaya
S
, et al
Telomere length and telomerase complex mutations in pediatric acute myeloid leukemia
.
Leukemia
2013
;
27
:
1786
9
.
46.
Nagel
I
,
Szczepanowski
M
,
Martin-Subero
JI
,
Harder
L
,
Akasaka
T
,
Ammerpohl
O
, et al
Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies
.
Blood
2010
;
116
:
1317
20
.
47.
Chien
WW
,
Catallo
R
,
Chebel
A
,
Baranger
L
,
Thomas
X
,
Bene
MC
, et al
The p16(INK4A)/pRb pathway and telomerase activity define a subgroup of Ph+ adult Acute Lymphoblastic Leukemia associated with inferior outcome
.
Leukemia Res
2015
;
39
:
453
61
.
48.
Kolquist
KA
,
Ellisen
LW
,
Counter
CM
,
Meyerson
M
,
Tan
LK
,
Weinberg
RA
, et al
Expression of TERT in early premalignant lesions and a subset of cells in normal tissues
.
Nature Genetics
1998
;
19
:
182
6
.
49.
Cameron
BJ
,
Gerry
AB
,
Dukes
J
,
Harper
JV
,
Kannan
V
,
Bianchi
FC
, et al
Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells
.
Sci Transl Med
2013
;
5
:
197ra03
.
50.
Lee
DW
,
Gardner
R
,
Porter
DL
,
Louis
CU
,
Ahmed
N
,
Jensen
M
, et al
Current concepts in the diagnosis and management of cytokine release syndrome
.
Blood
2014
;
124
:
188
95
.
51.
Straathof
KC
,
Pule
MA
,
Yotnda
P
,
Dotti
G
,
Vanin
EF
,
Brenner
MK
, et al
An inducible caspase 9 safety switch for T-cell therapy
.
Blood
2005
;
105
:
4247
54
.
52.
Di Stasi
A
,
Tey
SK
,
Dotti
G
,
Fujita
Y
,
Kennedy-Nasser
A
,
Martinez
C
, et al
Inducible apoptosis as a safety switch for adoptive cell therapy
.
N Engl J Med
2011
;
365
:
1673
83
.