Cancer
Research
Microenvironment and Immunology
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
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 BCLL 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.
Introduction
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 TILbased 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 na€ve 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
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-theshelf" reagents to treat a large spectrum of tumors (1). At present,
three types of ACT protocols can be defined based on isolated
1
Department of Medicine, Section of Immunology, University of Verona, Verona, Italy. 2Familial Cancer Clinic and Oncoendocrinology,
Veneto Institute of Oncology, Padova, Italy. 3Department of Surgery,
Loyola University Medical Center, Maywood, California. 4CESI Aging
Research Center, G. D'Annunzio University, Chieti Scalo, Chieti, Italy.
5
Department of Computer Science, University of Verona,Verona, Italy.
6
Department of Neurological and Movement Sciences, University of
Verona, Verona, Italy. 7Department of Pulmonary Medicine, Erasmus
MC, Rotterdam, the Netherlands. 8University of Verona, Interdepartmental Laboratory for Medical Research (LURM), Verona, Italy.
9
Department of Medicine, Section of Hematology, University of Verona, Verona, Italy.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Vincenzo Bronte and Stefano Ugel contributed equally to this article.
Corresponding Author: Vincenzo Bronte, University Hospital and Department
of Medicine, Immunology Section, Verona, P.le L.A. Scuro, 10, Verona, VR 37134,
Italy. Phone: 39-045-8124007; Fax: 39-045-8126455; E-mail:
[email protected]
doi: 10.1158/0008-5472.CAN-15-2318
2016 American Association for Cancer Research.
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Anti-Telomerase Adoptive T-cell Therapy
approach that was recently improved by the immunization of
human antigen–negative mice engineered to bear the whole
human TCR-a and b 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 (BCLL). 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.
Materials and Methods
Mice
C57BL/6 (C57BL/6NCrl) mice were purchased from Charles
River Laboratories Inc.; OT-1 (C57Bl/6-Tg(TcraTcrb)1100Mjb/
J) and CD45.1þ mice (B6.SJL-PtrcaPepcb/BoyJ) from The Jackson Laboratory; NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac)
and Rag2//g c/ mice (B10;B6-Rag2tm1Fwa II2rgtm1Wjl) from
Taconic. The transgenic mice IgH.TEm 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) CD34þ 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).
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Generation of hTERT865–873– and hHCV1406–1415–specific, TCRtransduced 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 mg/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 CD4þ and CD8þ 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 CD8þ 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 mg/mL LPS maturation,
pulsed with 10 mg/mL of the specific peptide. DCs were then used
to stimulate T cells at an E/T ratio of 10:1 in complete RPMI1640 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. CD8þ T cells
were screened for hTERT865–873 dextramer positivity and
hTERT865–873–specific reactivity in IFNg ELISA.
Systemic treatment of mouse leukemic chimeras
Chimeras were generated combining 106 CD45.2þ IgH.TEm
and 4 106 CD45.1þ syngeneic WT BM cells in Rag2/gc/
mice, after preconditioning with Busulfan (25 mg/kg). When
CD45.2þ cells raised to 15% of total B cells, 5 106
mTERT198–205– or OVA257–264–specific CTLs were intravenously
injected twice in mice after g-irradiation, followed by recombinant IL2 administration (17).
Systemic treatment of human leukemic chimeras
NOG mice were g-irradiated (1.20 Gy) and subsequently
engrafted with 105 human HLA-A2CD34þ 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 meanSD. 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.
Results
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
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Sandri et al.
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
þ
(CTRL : 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.
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.TEm mouse model in which the
sporadic SV40 large T antigen (hereafter indicated as SV40þ)
expression in mature B cells generates a B-CLL–like neoplasia
(32). We considered mice as a leukemic (defined henceforward as
IgH.TEmþ) when the majority of CD19þ B cells expressed the IgMb
allele (IgMb > 75%). Splenic B cells isolated from IgH.TEmþ 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.TEm or WT mice. Only B cells
purified from IgH.TEmþ mice were efficiently recognized by polyclonal mTERT198–205 CTLs, both in a cytofluorimetric cytotoxic
assay (summarized in Supplementary Fig. S2A) and IFNg 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.
The low incidence of leukemia did not allow us to verify in vivo
the therapeutic efficacy of a TERT-based ACT in IgH.TEm mice, so
we engrafted BM cells isolated from an IgH.TEmþ mouse into
immunodeficient Rag-2/g c/ 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.TEmþ 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 SV40þ
CD19þCD5þB220low/ cells, similar to what occurred in the
2542 Cancer Res; 76(9) May 1, 2016
donor IgH.TEmþ 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 (CD45.2þ) derived from IgH.TEmþ mouse (as
previously described) and four-fifths of normal congenic BMcells (CD45.1þ) isolated from WT mice, into immunodeficient
Rag-2/g c/ mice. When mice had about 15% of CD19þ
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 CD19þ B lymphocytes
compared with control mice (Fig. 2A). However, mTERT-based
ACT did not affect the normal B-cell reconstitution, because the
levels of CD19þ 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 CD19þCD45.2þ leukemic cell expansion without influencing the normal CD19þCD45.1þ 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 mTERTspecific ACT promoted a significant contraction in splenic SV40þ
cells compared with control ACT, which was associated with
an increased number of normal B220þ cells (Fig. 2D). Flow
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Anti-Telomerase Adoptive T-cell Therapy
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
þ
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
þ
þ
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 CD19 CD45.2
þ
þ
B cells (mTERT vs. OVA ACT, P ¼ 0.013; RM ANOVA test, left) and normal CD19 CD45.1 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
þ
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
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
overall percentage of splenic CD19þ cells was not modified by
mTERT-specific ACT, the relative ratio between malignant
CD45.2þ cells and healthy CD45.1þ cells significantly changed
(Fig. 2E). As expected from this anti-leukemic activity, mTERTspecific ACT significantly improved the leukemia-bearing mouse
survival (Fig. 2F).
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-A2þ HDs. We isolated CD3þ 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
CD8þ 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 CD8þ T cells (Fig. 3A). Patients whose T cells
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displayed positivity for hTERT-specific dextramer staining were
also able to release IFNg 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.
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 a
and b chains of the TCR (Fig. 3D) were then cloned by Va- and Vbspecific primer panels, amplified by PCR and the products of the
reaction were sequenced and inserted into a retroviral vector (34)
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–specific TCR
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.
vitro transduced with either anti-hTERT or anti-hHCV (as control)
TCR-encoding retroviruses (36), we checked the expression of the
mouse TCR Vb chain (mVb3), 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
2544 Cancer Res; 76(9) May 1, 2016
recognized HLA-A2þ 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 CD4þ and CD8þ T lymphocytes engineered with
hTERT865–873–specific TCR showed an effector memory phenotype (CD45RACD62LCCR7; Supplementary Fig. S4A)
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Anti-Telomerase Adoptive T-cell Therapy
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
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, CD4þ T cells were polarized
toward IFNg-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-A2þ B-cell
tumor cell lines with the expected exception of HLA-A2 MEC-1
(Fig 4B). The broad antitumor therapeutic activity of
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hTERT865–873–specific, TCR-engineered T cells was confirmed in
vivo in immunodeficient NOG mice engrafted s.c. with five different HLA-A2þ 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.
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
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Sandri et al.
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.
after the first treatment, we assessed tumor spreading to different
organs (Fig. 5B–C). Moreover, we performed IHC of human
CD20þ cells in the spleen of ACT-treated mice (Fig. 5D). All these
analyses showed that engineered hTERT865–873–specific, TCRengineered T cells induced a significant reduction in human BCLL accumulation.
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-A2þ 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-A2þ 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 CD34þ cells, were
engrafted with either HLA-A2þ 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-A2þ PBMCs from B-CLL
patients, the total number of circulating hTERT865–873–specific,
TCR-engineered T cells inversely correlated with the percentage of
CD19þHLA-A2þ malignant B cells (Fig. 6B, left). On the contrary,
ACT with hTERT865–873–specific, TCR-engineered T cells had no
2546 Cancer Res; 76(9) May 1, 2016
significant effects on the number of hCD19þ 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-A2þ
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 CD8þ
T-cell infiltration, as measured by flow cytometry (data not
shown).
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 hTERTbased immunotherapeutic approach toward the hematopoietic
compartment. Although human CD34þHLA-A2þ 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 g-irradiated, immunodeficient NOG mice (Fig. 7C). Moreover, we treated human immune reconstituted (HIR) mice,
obtained by transplantation of human HLA-A2þ CD34þ cells
Cancer Research
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Anti-Telomerase Adoptive T-cell Therapy
Figure 6.
hTERT865–873–specific, TCR-engineered
þ
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, TCRengineered as assayed both by flowcytometry 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
þ
cells (n ¼ 20; CTRL ); HLA-A2 HD B
cells (n ¼ 20); HLA-A2 B cells from Bþ
CLL patients (n ¼ 10); HLA-A2 B cells
from B-CLL patients (n ¼ 10). ANOVA
test. B, humanized NOG mice,
challenged with PBMCs isolated from
þ
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 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-specific ACT. Bars,
100 mm; Student t test. D, as indicated in
C, evaluation of HLA-A2 leukemic cells
after hTERT- or hHCV-specific ACT. Bars,
100 mm. Student t test.
into immunodeficient NOG mice, with two consecutive ACTs
(Supplementary Fig. S5C). Seventeen weeks after CD34þ cell
engraftment, the total number of human CD45þ 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 (CD45þCD33þSSChigh and CD45þ
CD33þSSClow, 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 CD45þCD11bþCD16þ 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., CD45þ
CD11bþCD16þ, 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
www.aacrjournals.org
common consequence of conventional chemotherapeutic
treatments.
Discussion
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
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2547
Sandri et al.
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, HLAþ
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
þ
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.
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
þ
þ
cells. Data are from 1 of 2 independent experiments (n ¼ 6). Statistical analysis was performed with RM ANOVA: CD34 /hTERT ACT vs. CD34 /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 percentages of
þ
high
þ
low
granulocytes and CD33 /SSC monocytes (right). G, relative proportions of human cell populations in BM (left), with analysis of myeloid
CD33 /SSC
subpopulations (right). H, relative proportion of human BM granulocytes divided according to their maturation stages with CD11b and CD16 markers. Student
þ
t test. I, human BM cells derived from healthy donors were divided by FACS sorting in three main populations: CD11b CD16 (orange), CD11b CD16 (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.
2548 Cancer Res; 76(9) May 1, 2016
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Anti-Telomerase Adoptive T-cell Therapy
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 107 mol/L, which is modest compared with the TCR
affinity of our engineered T cells, which displayed 50% of their
maximum response at 1014 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.TEm 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-theshelf" 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 Vb3 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).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
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
Acknowledgments
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.
Grant Support
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.
Received August 24, 2015; revised January 5, 2016; accepted January 24, 2016;
published online May 2, 2016.
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