Feasibility of Telomerase-Specific Adoptive T-Cell Therapy for B-Cell Chronic Lymphocytic Leukemia and Solid Malignancies

Cancer Microenvironment and Immunology Research

Feasibility of Telomerase-Speciﬁc Adoptive T-cell Therapy for B-cell Chronic Lymphocytic Leukemia and Solid Malignancies Sara Sandri1, Sara Bobisse2, Kelly Moxley3, Alessia Lamolinara4, Francesco De Sanctis1, Federico Boschi5, Andrea Sbarbati6, Giulio Fracasso1, Giovanna Ferrarini1, Rudi W. Hendriks7, Chiara Cavallini8, Maria Teresa Scupoli8,9, Silvia Sartoris1, Manuela Iezzi4, Michael I. Nishimura3, Vincenzo Bronte1, and Stefano Ugel1

Abstract

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

Introduction tumor-infiltrating T cells (TIL), T-cell receptor (TCR) engineered T cells, or chimeric antigen receptor (CAR) transduced lymphocytes, The development of adoptive cell therapy (ACT) represents an all of them already tested in clinical settings (2). TIL-based ACT emerging and realistic approach to treat cancer patients. This is can result in a long-lasting and complete cancer regression in testified by the numerous phase II clinical trials, the approval of metastatic melanoma patients (3, 4). However, this approach specific T-cell therapies by the FDA, and the growing interest of remains a personalized treatment that displays several technical biotechnology and pharmaceutical industry to generate "off-the- constraints (5). The clinical response following adoptive TIL shelf" reagents to treat a large spectrum of tumors (1). At present, transfer was associated with T cells reactive toward mutated three types of ACT protocols can be defined based on isolated 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 1Department of Medicine, Section of Immunology, University of Ver- on genetically engineered lymphocytes where the desired TCR ona, Verona, Italy. 2Familial Cancer Clinic and Oncoendocrinology, € 3 sequence insertion, by a virus-mediated delivery into na ve T cells, Veneto Institute of Oncology, Padova, Italy. Department of Surgery, fi – Loyola University Medical Center, Maywood, California. 4CESI Aging can confer an antigen-oriented immune speci city (7 9). To Research Center, G. D'Annunzio University, Chieti Scalo, Chieti, Italy. develop rapidly and apply ACT to a wide range of human 5Department of Computer Science, University of Verona,Verona, Italy. 6 neoplastic diseases, the characterization of high-avidity TCRs that Department of Neurological and Movement Sciences, University of fi Verona, Verona, Italy. 7Department of Pulmonary Medicine, Erasmus ef ciently and broadly recognize cancer cells is thus a primary goal MC, Rotterdam, the Netherlands. 8University of Verona, Interdepart- (10). However, antitumor CTLs with a high-avidity TCR against mental Laboratory for Medical Research (LURM), Verona, Italy. non-mutated tumor-associated antigens (TAA) are normally 9 Department of Medicine, Section of Hematology, University of Ver- deleted during thymus education of self-reactive T cells (11), and ona, Verona, Italy. isolation of TCR recognizing individual mutations of patients' Note: Supplementary data for this article are available at Cancer Research cancers is feasible in theory (12) but currently not applicable to Online (http://cancerres.aacrjournals.org/). large scale, standardized therapy. Nowadays, high-avidity TCR Vincenzo Bronte and Stefano Ugel contributed equally to this article. sequences could be achieved by different approaches. T cells with Corresponding Author: Vincenzo Bronte, University Hospital and Department higher functional avidity could be generated in vitro by stimula- of Medicine, Immunology Section, Verona, P.le L.A. Scuro, 10, Verona, VR 37134, tion with autologous dendritic cells (DC) transfected with RNA Italy. Phone: 39-045-8124007; Fax: 39-045-8126455; E-mail: encoding an allogeneic major histocompatibility complex [email protected] (MHC) and the desired TAA (13). Alternatively, TCR can be doi: 10.1158/0008-5472.CAN-15-2318 isolated from mouse CTLs primed in vivo by vaccination of 2016 American Association for Cancer Research. transgenic mice bearing human HLA-A2 molecules (14, 15), an

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approach that was recently improved by the immunization of Generation of hTERT865–873– and hHCV1406–1415–specific, TCR- human antigen–negative mice engineered to bear the whole transduced T cells human TCR-a and b gene loci together with the HLA-A2 allele OKT-3–activated PBMCs were infected with the viral superna- (16). We previously reported the feasibility to isolate and enrich a tant of the hTERT865–873/PG13 cell line in the presence of hIL15 polyclonal T-cell population specific for human telomerase (100 mg/mL) and rIL2 (300 IU/mL; ref. 34). PBMCs were then (hTERT)865–873 epitope through in vitro stimulation of mouse T immunomagnetically enriched for CD34 (Miltenyi) and expand- lymphocytes isolated from HLA-A2.1 transgenic mice (17). These ed. Control HCV1406–1415 (KLVALGINAV)–specific, TCR-trans- CTLs recognized different hTERT-expressing human cancer cell duced T cells were generated following the same protocol. The þ þ lines, as well as colon cancer stem cells (17). Telomerase is percentage of CD4 and CD8 T cells (usually 20% and 80%, reactivated in the majority of human tumors independently of respectively) was always tested before in vitro or in vivo studies. In their histology (18), and several hTERT epitopes, which are general, the amount of T cells used for in vivo treatments were þ naturally processed and presented in association with MHC adjusted in order to inject 2.5 106 CD8 cells. molecules on tumor cell surface, have been already documented (19–22). It is thus not surprising that TERT was ranked among the Generation and expansion of telomerase-specific T cells from most prioritized TAAs (23), and several active immunotherapeu- B-CLL and HD PBMCs tic approaches based on TERT antigen have been exploited to B-CLL patients and HD were selected for HLA-A2 status, as target, both in vivo and in vitro, either autologous or allogeneic assessed by FACS. T cells were immunomagnetically isolated from antigen-presenting cells (APC), including antigenic peptides PBMCs. Human DCs were generated from CD14-selected mono- (24–27), RNA-based vaccines (28), as well as plasmid or viral cytes (Miltenyi Biotec) and, after 100 mg/mL LPS maturation, vectors encoding hTERT (29). Unfortunately, clinical responses in pulsed with 10 mg/mL of the specific peptide. DCs were then used these trials were limited, suggesting the need for more powerful, to stimulate T cells at an E/T ratio of 10:1 in complete RPMI- immune-based strategies. In the current study, we show the 1640 in presence of IL7 (10 ng/mL), IL15 (2 ng/mL), and IL2 feasibility to transduce human T cells with a high-avidity mouse (10 IU/mL; all from Miltenyi Biotec). At days 7 and 15 of culture, þ TCR able to recognize hTERT865–873 peptide in association with T cells were restimulated with peptide-pulsed DCs. CD8 T cells HLA-A2 molecules to control human solid tumors and hemato- were screened for hTERT865–873 dextramer positivity and logic malignancies, such as chronic lymphocytic leukemia (B- hTERT865–873–specific reactivity in IFNg ELISA. CLL). The high levels of hTERT in leukemic B cells correlated with poor clinic outcome (30, 31). We show here that hTERT-based Systemic treatment of mouse leukemic chimeras þ ACT can selectively eliminate leukemic B cells, causing a minor Chimeras were generated combining 106 CD45.2 IgH.TEm þ toxicity against normal myeloid cells, making this approach and 4 106 CD45.1 syngeneic WT BM cells in Rag2 / gc / suitable to clinical translation. mice, after preconditioning with Busulfan (25 mg/kg). When þ CD45.2 cells raised to 15% of total B cells, 5 106 Materials and Methods mTERT198–205– or OVA257–264–specific CTLs were intravenously Mice injected twice in mice after g-irradiation, followed by recombi- C57BL/6 (C57BL/6NCrl) mice were purchased from Charles nant IL2 administration (17). River Laboratories Inc.; OT-1 (C57Bl/6-Tg(TcraTcrb)1100Mjb/ þ J) and CD45.1 mice (B6.SJL-PtrcaPepcb/BoyJ) from The Jack- Systemic treatment of human leukemic chimeras son Laboratory; NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac) NOG mice were g-irradiated (1.20 Gy) and subsequently / / tm1Fwa tm1Wjl þ and Rag2 /gc mice (B10;B6-Rag2 II2rg )from engrafted with 105 human HLA-A2 CD34 cells via tail-vein Taconic. The transgenic mice IgH.TEm have been described injection, as previously reported (35). Mice were then injected previously (32), and their natural history of malignant pro- with 108 freshly isolated B-CLL PBMCs into retro-orbital plexus. gression has been monitored (Supplementary Fig. S1) in 6 Treatments with 2.5 10 hTERT865–873– or hHCV1406–1415– about 150 mice. All animal experiments were approved by the specific, TCR-transduced T lymphocytes were given 3 times, Verona University Ethical Committee, authorized by Ministe- weekly, starting 1 week after the engraftment of the pathology rial Decree (16/2014-B) and conducted according to the guide- and were always followed by IL2 administration. At sacrifice, lines of the Federation of European Laboratory Animal Science organs were collected and analyzed by flow cytometry and IHC. Associations. Statistical analysis Cell lines and patient samples Data were indicated as the meanSD. The Student t test was All cell lines were obtained from ATCC and were passaged for used to determine statistically significant differences between two fewer than 6 months after their purchase. Human cell line identities treatment groups, while the ANOVA test was used in case of were verified using short tandem repeat profiling. Healthy donor þ multiple comparisons. Growth curves were analyzed with repeat- (HD) CD34 cells derived from bone marrow (BM) were pur- ed-measures (RM) ANOVA. Survival analysis was performed chased from Lonza. Peripheral blood mononuclear cells (PBMC) using the Kaplan–Meier survival analysis (log-rank) method. All isolated from B-cell chronic lymphocytic leukemia (B-CLL) P values less than 0.05 were considered statistically significant. 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 com- Results pliance with the Declaration of Helsinki. The study was approved mTERT198–205–specific CTLs control mouse B-CLL progression by the local ethics committee (AOUI of Verona, n. 1496). Selected After repeated ACTs with polyclonal mouse (m)TERT198–205– B-CLL patients had heterogeneous Binet clinical stages (33). specific CTLs to treat prostate cancers, we did not detect major side

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Figure 1. Malignant B-CLL cells isolated from IgH. þ TEm 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 ¼ 6; IgH. þ TEm , n ¼ 20; IgH.TEm , n ¼ 10). Data show representative samples. B, cell killing activity of mTERT198–205–specific CTLs was evaluated by flow cytometry cytotoxic assay (top) and IFNg secretion assay (top); positive control, mTERT198–205–pulsed, WT B cells (CTRLþ: n ¼ 6); negative control, OVA257–264–pulsed WT B cells (CTRL, n ¼ 6); WT B cells (n ¼ 6); IgH. þ TEm B cells (n ¼ 20); IgH.TEm B cells (n ¼ 10). ANOVA test.

þ effects toward normal cells and tissues with the exception of a donor IgH.TEm mouse (Supplementary Fig. S3A). Overall, B transient and reversible B-cell depletion in lymphoid organs (17). cells isolated from tumor-bearing chimeric mice showed high We therefore verified the therapeutic efficacy of mTERT198–205– levels of mTERT expression and activity (Supplementary Fig. S3B) specific CTL administration to restrain the expansion of mono- and were recognized by mTERT198–205–specific CTLs (Supple- clonal B cells, using the IgH.TEm mouse model in which the mentary Fig. S3C). Therefore, the pre-leukemic B cells in BM were þ sporadic SV40 large T antigen (hereafter indicated as SV40 ) able to give rise to a full B-CLL–like disease. expression in mature B cells generates a B-CLL–like neoplasia In order to assess the in vivo efficacy of mTERT198–205–specific (32). We considered mice as a leukemic (defined henceforward as CTLs in immune-competent mice, we generated mouse chimeras þ þ IgH.TEm ) when the majority of CD19 B cells expressed the IgMb by transplanting a mixture of BM cells, comprising one-fifth of þ þ þ allele (IgMb > 75%). Splenic B cells isolated from IgH.TEm mice leukemic cells (CD45.2 ) derived from IgH.TEm mouse (as displayed higher levels of TERT protein (Fig. 1A, top), as reported previously described) and four-fifths of normal congenic BM- þ for human B-CLL (31), together with an increased TERT enzy- cells (CD45.1 ) isolated from WT mice, into immunodeficient / / þ matic activity (Fig. 1A, bottom) in comparison with B lympho- Rag-2 gc mice. When mice had about 15% of CD19 cytes purified from either IgH.TEm or WT mice. Only B cells circulating cells, they were treated with two repeated transfers of þ purified from IgH.TEm mice were efficiently recognized by poly- either mTERT198–205– or the control, OVA257–264–specific CTLs clonal mTERT198–205 CTLs, both in a cytofluorimetric cytotoxic (Supplementary Fig. S3D). The leukemic mice treated with assay (summarized in Supplementary Fig. S2A) and IFNg release mTERT198–205–specific CTLs displayed a significant reduction in þ assay (Fig. 1B). These findings demonstrate that leukemic B the total number of blood circulating CD19 B lymphocytes lymphocytes can naturally process the endogenous TERT198–205 compared with control mice (Fig. 2A). However, mTERT-based peptide and present it in a MHC class I–restricted fashion. ACT did not affect the normal B-cell reconstitution, because the þ The low incidence of leukemia did not allow us to verify in vivo levels of CD19 B-cells in treated chimeras engrafted with only the therapeutic efficacy of a TERT-based ACT in IgH.TEm mice, so normal (WT) BM cells were not significantly different compared þ we engrafted BM cells isolated from an IgH.TEm mouse into with untreated mice (Fig. 2A). In fact, mTERT-based ACT selec- / / þ þ immunodeficient Rag-2 gc mice, partially ablated with che- tively reduced the CD19 CD45.2 leukemic cell expansion with- þ þ motherapy, to establish a standardized B-CLL–like pathology. In out influencing the normal CD19 CD45.1 B-cell development fact, all engrafted mice developed a B-CLL with the same features (Fig. 2B). This therapeutic effect of mTERT-specific ACT was þ of the IgH.TEm mouse donor and tumor-bearing mice had to be mirrored by the significant inverse correlation between the fre- euthanized 24 days after BM cell transplant (results for 2 passages quencies of blood circulating, mTERT198–205–specific CTLs with out of 9 total are shown). The transplants did not affect the the percentage of circulating B-CLL cells (Fig. 2C). The mTERT- þ leukemic phenotype: the histological structure of the spleen was specific ACT promoted a significant contraction in splenic SV40 þ almost completely replaced by a uniform infiltration with SV40 cells compared with control ACT, which was associated with þ þ þ CD19 CD5 B220low/ cells, similar to what occurred in the an increased number of normal B220 cells (Fig. 2D). Flow

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Figure 2. mTERT-based ACT controls mouse B-CLL progression. Leukemia-bearing chimeras were g-irradiated (200 cGy) and treated with two weekly ACTs based on mTERT198–205–specific(n ¼ 32) or OVA257–264–specific(n ¼ 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 CD19þ B cells (mTERT vs. OVA ACT, P ¼ 0.002; WT þ mTERT ACT vs. WT P ¼ 0.8; RM ANOVA test). B, flow cytometric evaluation of both circulating malignant CD19þCD45.2þ B cells (mTERT vs. OVA ACT, P ¼ 0.013; RM ANOVA test, left) and normal CD19þCD45.1þ B cells (mTERT vs. OVA ACT, P ¼ 0.14; RM ANOVA test, right). C, Spearman rank correlation between circulating, infused antigen-specific CTLs (anti-Vb5.2 for OVA257–264–specific CTLs and anti-Vb11 for mTERT198–205–specific CTLs, þ þ 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 mm. Data are mean SD of 1 of 4 independent experiments (n ¼ 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 CD19þ 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 þ þ malignant CD19 CD45.2 cells were 80% of total PBMCs. Kaplan–Meier analysis: mTERT ACT (n ¼ 32) versus OVA ACT (n ¼ 32), P < 0.001.

cytometry analysis on BM and spleen confirmed that, even if the displayed positivity for hTERT-specific dextramer staining were þ overall percentage of splenic CD19 cells was not modified by also able to release IFNg in response to hTERT865–873 peptide (Fig. mTERT-specific ACT, the relative ratio between malignant 3B), with a linear relationship between cytokine production and þ þ CD45.2 cells and healthy CD45.1 cells significantly changed the number of antigen-specific cells in culture (Fig. 3B, inset). (Fig. 2E). As expected from this anti-leukemic activity, mTERT- Then, we examined the association between the presence of specific ACT significantly improved the leukemia-bearing mouse endogenous anti-hTERT865–873 response and disease progression, survival (Fig. 2F). measured as time from diagnosis to initial therapy (time to first treatment, TTFT). Comparison between TTFT curves showed a Generation and characterization of engineered hTERT865–873– trend to a shorter TTFT in the "TERT nonresponder" group (Fig. specific T cells 3C). These data clearly unveiled the previously unknown presence To verify the presence of a TERT-specific immune response of endogenous T cells specific for a physiologically processed induced by B-CLL progression, we selected B-CLL patients, sharing hTERT865–873 epitope in B-CLL patients. HLA-A2 allele and clinical stage (according to the Binet staging We therefore explored the feasibility to redirect human T system), with at least 4-year follow-up; as a control, we included lymphocytes by genetic engineering based on de novo expression þ þ HLA-A2 HDs. We isolated CD3 T cells from patients and HD of a high-avidity, HLA-A2–restricted, hTERT-specific TCR. Briefly, PBMCs and cocultured them with either hTERT865–873– or we first isolated a mouse CTL clone expressing a TCR for human – hCMV495–503 peptide pulsed (as positive control of stimulation), HLA-A2–restricted hTERT865–873 epitope from a polyclonal, in in vitro differentiated, HLA-A2–matched DCs. After 3 weekly in vitro stabilized T cell population obtained from HLA-A2 trans- vitro stimulations, we verified the presence of hTERT-specific genic mice vaccinated toward hTERT (17). The sequences of the a þ CD8 T cells, defined as hTERT865–873 dextramer–positive cells, and b chains of the TCR (Fig. 3D) were then cloned by Va- and Vb- in approximately 50% of the B-CLL patients but not in HDs, even specific primer panels, amplified by PCR and the products of the though in almost all patients and HDs we detected the presence of reaction were sequenced and inserted into a retroviral vector (34) þ hCMV-specific CD8 T cells (Fig. 3A). Patients whose T cells able to transduce na€ve T cells. After expansion of human T cells in

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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 ¼ 8) and "TERT nonresponders" (n ¼ 8) according to a hTERT865–873 dextramer positivity greater than 0.02%. ANOVA test. Representative dot plots are shown. B, IFNg production after in vitro incubation of T cells isolated from either B-CLL patients or HDs. Columns show hIFNg 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 hIFNg 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–specificTCR sequence used to engineer human T cells. E, flow-cytometric analysis of mouse Vb3 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 IFNg 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.

þ vitro transduced with either anti-hTERT or anti-hHCV (as control) recognized HLA-A2 PBMCs and activated T-lymphocytes TCR-encoding retroviruses (36), we checked the expression of the (infected with retrovirally encoded anti-hHCV TCR) only when mouse TCR Vb chain (mVb3), a component of the hTERT865–873– the target T cells were pulsed with the hTERT865–873 peptide; no specific TCR, on T-cell surface. (Fig. 3E). We further confirmed the recognition of PBMCs and activated T lymphocytes, either presence of the correct, antigen-specific TCR on transduced T cells unpulsed or pulsed with the control hHCV1406–1415 peptide, by dextramer staining. (Fig. 3E). The engineered hTERT865–873 could be detected (Fig. 3G). These data indicate that –specific T cells displayed a much higher avidity TCR compared hTERT865–873-specific, TCR-engineered T lymphocytes cannot kill with the endogenous TCRs expressed by T lymphocytes isolated human proliferating T lymphocytes. from B-CLL patients, confirming the expected, intrinsic low avidity of the endogenous T-cell repertoire for self-antigens (Fig. 3F). Engineered hTERT865–873–specific, TCR-engineered T cells fi in vivo Finally, we verified that hTERT865–873–specific TCR-engineered T ef ciently restrain human solid tumor and B-CLL growth þ þ cells did not cause a catastrophic MHC-restricted T-cell fratricide, Both CD4 and CD8 T lymphocytes engineered with as recently shown for survivin-specific, TCR-engineered T lym- hTERT865–873–specific TCR showed an effector memory pheno- phocytes (37). The hTERT865–873–specific, TCR-engineered T cells type (CD45RA CD62L CCR7 ; Supplementary Fig. S4A)

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Figure 4. þ 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 51 hTERT865-873–specific, TCR-engineered T cells was evaluated by the Cr release assay (left) and hIFNg 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 ¼ 8) versus hHCV ACT (n ¼ 4); SW480, hTERT ACT (n ¼ 6) versus hHCV ACT (n ¼ 6); MDA-MB-231, hTERT ACT (n ¼ 7) versus hHCV ACT (n ¼ 7).

characterized by high expression of CD69/CD44/CD38/CD25 hTERT865–873–specific, TCR-engineered T cells was confirmed in and HLA-DR markers and low expression of exhaustion markers vivo in immunodeficient NOG mice engrafted s.c. with five dif- þ (LAG3/PD-1/Tim-3) compared with the specific isotype controls ferent HLA-A2 tumors. Both adoptively transferred þ (Supplementary Fig. S4C). Moreover, CD4 T cells were polarized hTERT865–873– and hHCV1406–1415–specific, TCR-transduced T toward IFNg-producing Th1 cells (Supplementary Fig. S4D). We cells comparably migrated to the tumor site (Fig. 4C), although selected several tumor cell lines of different histology, comprising only hTERT-specific ACT significantly controlled tumor growth hematologic malignancies such as myeloma (U266), B-CLL and prolonged mice survival (Fig 4D and Supplementary Fig. (JVM13 and MEC-1), and Burkitt lymphoma cells (DG-75), and S4E). These data clearly advocate the potential effectiveness of solid tumors, such as melanoma (SK23MEL), breast carcinoma TERT-based ACT approach to treat different human cancers. (MDA-MB-231), and colon carcinoma (SW480) cells. The select- We treated leukemic-bearing mice, systemically engrafted ed tumor cell lines showed high levels of TERT activity and with JVM13-Luc cells, for 3 consecutive weeks with either heterogeneous levels of both TERT protein and HLA-A2 mem- hTERT865–873– or hHCV1406–1415–specific, TCR-transduced T brane complex (Fig. 4A). The hTERT865–873–specific, TCR-engi- cells, weekly monitoring tumor cell expansion by biolumines- þ neered T cells were capable of recognizing in vitro HLA-A2 B-cell cence imaging. The hTERT-specific ACT induced a significant tumor cell lines with the expected exception of HLA-A2 MEC-1 decrease in radiance signals in leukemia-bearing mice compared (Fig 4B). The broad antitumor therapeutic activity of with mice receiving hHCV-specific ACT (Fig. 5A). Thirty-five days

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Figure 5. hTERT865–873–specific, TCR-engineered T cells restrain human B-CLL progression. A, therapeutic efficacy of hTERT-specific ACT þ in controlling JVM-13 Luc cell expansion (n ¼ 5 each; hTERT vs. hHCV ACT, P ¼ 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 þ evaluation of infiltrating hCD19 cells in different organs (BM, P < 0.001; spleen, P < 0.001; lung, P ¼ 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 mm. All data are mean SD of a representative experiment of two independent experiments. Student t test.

þ after the first treatment, we assessed tumor spreading to different significant effects on the number of hCD19 cells developed in organs (Fig. 5B–C). Moreover, we performed IHC of human mice engrafted with HLA-A2 B-CLL (Fig. 6B, right). One month þ CD20 cells in the spleen of ACT-treated mice (Fig. 5D). All these after B-CLL engraftment, we evaluated malignant B-cell infiltra- þ analyses showed that engineered hTERT865–873–specific, TCR- tion in both spleen and BM. The hTERT-specific ACT in HLA-A2 engineered T cells induced a significant reduction in human B- B-CLL–bearing mice visibly controlled the leukemia progression CLL accumulation. (Fig. 6C). Conversely, hTERT-specific ACT did not control the To further explore the usefulness of this immunotherapeutic neoplastic progression in HLA-A2 B-CLL–bearing mice (Fig. þ approach from a translational standpoint, we evaluated the 6D), although we detected the same amount of human CD8 hTERT presence in PBMCs isolated from B-CLL patients and T-cell infiltration, as measured by flow cytometry (data not age-matched HDs. PBMCs of B-CLL patients exhibited higher shown). hTERT expression and enzymatic activity levels compared with HD PBMCs (Supplementary Fig. S5A). Moreover, B cells isolated Adoptive transfer of hTERT865–873–specific, TCR-engineered T þ from HLA-A2 B-CLL patients were also recognized in vitro by cells induces the depletion of mature granulocytes but does not hTERT865–873–specific, TCR-engineered T lymphocytes similarly affect hematopoietic stem cells to the JVM13 cell line, while HLA-A2 B-CLL PBMCs as well as Even though engineered T cell–based therapies have shown þ HLA-A2 HD PBMCs were not recognized (Fig. 6A). We finally long-term efficacy and promising curative potential for the treat- tested the anti-leukemic activity of ACT against B-CLL patients' ment of cancer, several "on-target, off-tumor" toxicities have been PBMCs in vivo. To this aim, immunodeficient NOG mice, previ- reported (38). We therefore investigated the toxicity of hTERT- þ ously humanized with allogeneic BM-derived CD34 cells, were based immunotherapeutic approach toward the hematopoietic þ þ þ engrafted with either HLA-A2 or HLA-A2 PBMCs from B-CLL compartment. Although human CD34 HLA-A2 cells were dim- patients previously classified as TERT responders (Fig. 3C) and ly recognized by hTERT865–873–specific, TCR-engineered T cells treated with three weekly courses of ACT (Supplementary Fig. upon in vitro coculture (Fig. 7A), they maintained their in vitro þ S5B). In mice inoculated with HLA-A2 PBMCs from B-CLL ability to proliferate and differentiate into colonies in a semisolid patients, the total number of circulating hTERT865–873–specific, medium (Fig. 7B) and preserved their multipotency once injected TCR-engineered T cells inversely correlated with the percentage of into g-irradiated, immunodeficient NOG mice (Fig. 7C). More- þ þ CD19 HLA-A2 malignant B cells (Fig. 6B, left). On the contrary, over, we treated human immune reconstituted (HIR) mice, þ þ ACT with hTERT865–873–specific, TCR-engineered T cells had no obtained by transplantation of human HLA-A2 CD34 cells

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Figure 6. hTERT865–873–specific, TCR-engineered T cells selectively recognize HLA-A2þ 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 hIFNg release assay (bottom). Data are mean SD of three independent experiments: hTERT865–873–pulsed þ þ HLA-A2 HD B cells (n ¼ 20; CTRL ); þ hHCV1406–1415–pulsed HLA-A2 HD B cells (n ¼ 20; CTRL); HLA-A2þ HD B cells (n ¼ 20); HLA-A2 B cells from B- þ CLL patients (n ¼ 10); HLA-A2 Bcells from B-CLL patients (n ¼ 10). ANOVA test. B, humanized NOG mice, challenged with PBMCs isolated from B-CLL patients (HLA-A2þ patient, left; HLA-A2 patient, right), were treated with hTERT- or hHCV-specificACTs. Spearman rank correlation between circulating malignant B-cells and circulating infused engineered T cells (hTERT ACT, n ¼ 9; hHCV ACT, n ¼ 7; circles, evaluation after the second ACT; triangles, evaluation after the third ACT). C, IHC (left) and flow cytometric (right) þ evaluation of HLA-A2 leukemic cells after hTERT- or hHCV-specificACT.Bars, 100 mm; Student t test. D, as indicated in C, evaluation of HLA-A2 leukemic cells after hTERT- or hHCV-specificACT.Bars, 100 mm. Student t test.

into immunodeficient NOG mice, with two consecutive ACTs common consequence of conventional chemotherapeutic þ (Supplementary Fig. S5C). Seventeen weeks after CD34 cell treatments. þ engraftment, the total number of human CD45 cells in the fi – spleen was not affected by hTERT-speci c ACT (Fig. 7D E), while Discussion they were significantly reduced in the BM compared with mice receiving control ACT (Fig. 7E). The relative distribution among We show here the strength and safety of a broadly applicable human leukocytes was maintained in the spleen (Fig. 7F, left), as immunotherapy protocol based on engineered T cells generated well as the percentage of both human granulocytes and mono- by transduction with a high-affinity TCR capable of recognizing þ þ high þ cytes among myeloid cells (CD45 CD33 SSC and CD45 the complex formed by hTERT865–873 peptide and HLA-A2 mol- þ CD33 SSClow, respectively; Fig. 7F, right). Moreover, we could ecule. Telomerase can be regarded as a universal TAA: it contri- not identify any alteration of relative distribution of myeloid and butes to sustain tumor cell survival (40) and prevent apoptosis lymphoid cells in BM (Fig. 7G, left), except for a significant elicited by antiproliferative agents (41). Furthermore, hTERT is contraction in human granulocytes (Fig. 7G, right). Indeed, immunogenic (27) and, indeed, five HLA-A2–restricted epitopes among granulocytic cell subsets, we observed a nearly complete have already been identified: I540 (19), R865 (20), 572Ya and þ þ þ deletion of the more mature CD45 CD11b CD16 human 988Ya (42), and R38a (22). The first identified peptide, I540, was myeloid cells in mice treated with hTERT-specific ACT also tested in different cancer vaccination clinical trials that (Fig. 7H). To confirm a potential hTERT-specific, ACT-linked showed a modest impact on tumor control (26, 27). To the best toxicity on myeloid mature cell subsets, we purified from human of our knowledge, we show here for the first time that a specific BM aspirate of HDs the three main granulocytic maturation cohort of B-CLL patients, with a trend toward a less aggressive cell fractions (myeloblasts, promyelocytes, and granulocytes; leukemia, presented a specific, endogenous response toward the þ ref. 39). Only the more mature cell population, i.e., CD45 R865 epitope that was not detectable in HDs (Fig. 3A–B). Our þ þ CD11b CD16 , was significantly recognized in vitro by data diverge from results published about I540 epitope, which is hTERT865–873–specific, TCR-engineered T cells (Fig. 7I), although able to promote a comparable immune response in both tumor no difference in telomerase enzyme activity levels was detected patients and normal donors (43). The differences between I540 among the different cell fractions (data not shown). Thus, and R865 epitopes might be related to a different baseline of hTERT865–873–specific, TCR-engineered T cells ignore progenitors immune tolerance between the two epitopes. Moreover, the TCR fi and specifically target only mature myeloid subsets, potentially af nity of the endogenous anti-hTERT865–872 T cells isolated from limiting the induction of a prolonged adverse neutropenia, a B-CLL patients is deeply low (Fig. 3F), which makes it unrealistic

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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- þ þ þ 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 hIFNg were evaluated by ELISA. Data are mean SD of three independent experiments. ANOVA test. B, after incubation, isolated hCD34þ cells were used in a colony-forming unit (CFU) assay. Data are mean SD of three independent experiments. Student t test. þ þ 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 ¼ 6). Statistical analysis was performed with RM ANOVA: CD34þ/hTERT ACT vs. CD34þ/hHCV ACT, þ P ¼ 0.32. D, distribution of human CD45 leukocytes in the spleen of humanized NOG mice treated with hTERT865–873– (n ¼ 7) or control HCV1406–1415– (n ¼ 6) specific, TCR-engineered T cells. Bars, 50 mm. Data are mean SD of 1 of 2 independent experiments. Student t test, P ¼ 0.64. E, flow cytometric analysis of þ hCD45 splenocytes and BM cells isolated from ACT-treated HIR mice. F, relative proportions of human splenic leukocytic populations (left) and percentagesof CD33þ/SSChigh granulocytes and CD33þ/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), CD11bþCD16 (green), and þ þ 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. IFNg levels were assessed by ELISA. Bars, 50 mm. Data are mean SD of four experiments. ANOVA test.

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to isolate high-avidity T cells from patients to develop an ACT case of unfavorable events (51). The clinic impact of these new protocol. So far, only one study reported the redirected T cell– strategies was recently demonstrated to control the graft versus based adoptive immunotherapy targeting hTERT with a TCR host disease (GVHD) symptoms in acute leukemia relapsed isolated from human CTLs (21), generated from PBMCs of HDs patients, after allogeneic stem-cell transplant (52). Importantly, in vitro stimulated with HLA-A24:02–restricted nonameric we also excluded a fratricide effect of engineered T cells, as well as hTERT461–469 peptide (44). However, the overall functional avid- the elimination of activated endogenous T cells, which might ity to hTERT461–469 peptide evaluated for transduced T cells was limit the power of immunotherapy. Finally, for a clinical point of about 10 7 mol/L, which is modest compared with the TCR view, it could be feasible to isolate a fully humanized TCR against affinity of our engineered T cells, which displayed 50% of their hTERT by immunization of antigen-negative humanized mice maximum response at 10 14 mol/L peptide concentration (Fig. that can generate optimal affinity TCRs for T-cell therapy (16). 3F). Transduced anti-hTERT T cells specifically target tumor cells, such as leukemic B cells both in vitro and in vivo, without affecting Disclosure of Potential Conflicts of Interest normal B-cell development. Indeed, anti-TERT T cells do not alter No potential conflicts of interest were disclosed. dramatically stem cell differentiation, suggesting a lower toxicity compared with common chemotherapies that normally promote Authors' Contributions severe myeloid precursor depletion in treated patients, often Conception and design: S. Sandri, C. Cavallini, M.T. Scupoli, S. Sartoris, requiring the administration of support therapy to restore normal V. Bronte, S. Ugel Development of methodology: S. Sandri, G. Fracasso, R.W. Hendriks, S. Ugel hematopoiesis. The ability of TERT targeting to control B-CLL fi Acquisition of data (provided animals, acquired and managed patients, progression is also con rmed by our data using IgH.TEm mice; in provided facilities, etc.): S. Sandri, F. De Sanctis, F. Boschi, G. Ferrarini, fact, polyclonal anti-TERT CTLs transfer was able to significantly C. Cavallini, M.T. Scupoli, M.I. Nishimura improve the leukemia-bearing mouse survival (Fig 2I). The ability Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Sandri, A. Lamolinara, F. De Sanctis, F. Boschi, of hTERT865–873–specific, TCR-engineered T cells to eliminate mature granulocytes hints at an extension of this therapy toward 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, human acute myeloid leukemia (AML), that is characterized by a V. Bronte, S. Ugel strong TERT activity directly correlating with poor outcome of the Study supervision: S. Sartoris, V. Bronte disease (45). Another potential target for hTERT-specific ACT is Other (performed research and provided essential new reagents): S. Bobisse, represented by B-cell acute lymphoblastic leukemia, in which K. Moxley TERT locus is recurrently targeted by somatic chromosomal Other (assisted with experimental design of in vivo imaging study): translocations (46) and TERT expression is a marker associated A. Sbarbati Other (analyzed tissue sections by immunohistochemistry and interpreted with inferior clinical outcome (47). Finally, our data about the the data): A. Lamolinara therapeutic ability of hTERT-specific ACT to efficiently control cancer progression of different solid tumors (Fig. 4B) suggest how Acknowledgments the transduced anti-hTERT T cells could be a potential "off-the- The authors thank Elisa Zoratti, Mauro Giacca, Lorena Zentilin, Martina shelf" reagent applicable to treat many oncologic diseases. Tinelli, Loredana Ruggeri, Ornella Poffe, Rosalinda Trovato, Alessandra Fiore, TERT is physiologically activated in a limited number of human and Cristina Anselmi for technical help. normal cell populations (48). Unfortunately, experimental models that can predict potential toxic effects against these human cells Grant Support are currently not available and some off-target activity can be This work was supported by grants from the Italian Ministry of Health, Italian completely unpredictable (49). However, to control off-target Ministry of Education, Universities, and Research (FIRB cup: B31J11000420001), Italian Association for Cancer Research (AIRC; grants 6599, 12182, and 14103), activity and mitigate excessive in vivo T-cell activation/expansion National Cancer Institute (P01 CA154778 to M.I. Nishimura), and Dutch Cancer after systemic infusion, which might induce a lethal cytokine Society (R.W. Hendriks). storm (50), we plan to develop an antidote based on the admin- The costs of publication of this article were defrayed in part by the payment of istration of antibodies specific for the mouse Vb3 chain of the page charges. This article must therefore be hereby marked advertisement in engineered TCR. Moreover, to limit direct toxicity, uncontrolled accordance with 18 U.S.C. Section 1734 solely to indicate this fact. growth and malignant transformation of hTERT865–873–specific, TCR-engineered T cells, we plan to insert a suicide gene, such as the Received August 24, 2015; revised January 5, 2016; accepted January 24, 2016; inducible caspase-9 gene (iCasp9), which can be triggered in the published online May 2, 2016.

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