Barbara Savoldo 1, 5 *, Cliona M Rooney 1, 2, 5, Antonio Di Stasi 1, Hinrich Abken 3, Andreas Hombach 3, Aaron E Foster 1, Lan Zhang 1, Helen E Heslop 1, 4, 5, Malcolm K Brenner 1, 4, 5, and Gianpietro Dotti 1, 4
1 Center for Cell and Gene Therapy, Baylor College of
Medicine, The Methodist Hospital and Texas Children's Hospital, Houston,
TX, United States
2 Department of Molecular Virology and Microbiology,
Baylor College of Medicine, The Methodist Hospital and Texas Children's
Hospital, Houston, TX, United States
3 Tumor Genetics, University Hospital Cologne, Cologne,
Germany
4 Department of Medicine, Baylor College of Medicine,
The Methodist Hospital and Texas Children's Hospital, Houston, TX, United
States
5 Department of Pediatrics, Baylor College of Medicine,
The Methodist Hospital and Texas Children's Hospital, Houston, TX, United
States
* Corresponding author; Phone: +1 832-824-4725; Fax:
+1 832-825-4732
email: bsavoldo@bcm.tmc.edu
Adoptive transfer of Epstein Barr Virus (EBV)-specific cytotoxic T-lymphocytes (EBV-CTLs) has shown that these cells expand and persist in patients with EBV+ Hodgkin's lymphoma (HD), to produce complete tumor responses. Treatment failure, however, occurs if a subpopulation of malignant cells in the tumor lacks or loses expression of EBV-antigens. We have therefore determined whether we could prepare EBV-CTLs that retained the anti-tumor activity conferred by their native receptor whilst expressing a chimeric antigen receptor (CAR) specific for CD30, a molecule highly and consistently expressed on malignant Hodgkin Reed-Sternberg cells. We made a CD30CAR and were able to express it on 26%±11% and 22%±5% of EBV-CTLs generated from healthy donors and HD patients, respectively. These CD30CAR+ CTLs killed both autologous EBV+ cells through their native receptor and EBV-/CD30+ targets through their MHC-unrestricted CAR. A subpopulation of activated T-cells also express CD30, but the CD30CAR+ CTLs did not impair cellular immune responses, likely because normal T-cells express lower levels of the target antigen. In a xenograft model, CD30CAR+ EBV-CTLs could be co-stimulated by EBV-infected cells and produce anti-tumor effects even against EBV-/CD30+ tumors. EBV-CTLs expressing both a native and a chimeric antigen receptor may therefore have added value for treatment of HD.
Chemotherapy and radiotherapy cures more than 80% of patients
with Hodgkin’s
lymphoma (HD) [1,2] . However, a subset
of patients have primary resistant disease, or relapse
even after high-dose chemotherapy and autologous stem cell transplantation
[3]. Alternative
therapeutic strategies are thus required to treat patients with
resistant/relapsed disease, as well
as to reduce the morbidity attributable to chemo/radiotherapy
[4].
In almost 40% of HD patients, Hodgkin Reed Sternberg (HRS)
tumor cells express
Epstein Barr virus (EBV) associated antigens [5]
, and we have shown that the adoptive transfer of
EBV-specific cytotoxic T cells (EBV-CTLs) is well tolerated
and can induce disease responses
including remission [6,7], so that in principle
immunotherapy could be an alternative treatment for
this disease. However, HD tumor cells lack expression of immunodominant
EBV antigens, and
only three weakly immunogenic antigens (EBNA1, LMP1 and LMP2) can
be detected [5] . In
response to CTL therapy, subpopulations of EBV+ HD tumor cells may
lack or lose expression
of these weak antigens, allowing tumor escape and consequent treatment
failure.
Almost all HRS cells overexpress the CD30 molecule, which is a member
of the tumor
necrosis factor family [8] . Clinical trials of
CD30 monoclonal antibodies (MAbs) (conjugated with
immunotoxins [9,10] or radioisotopes [11]
) produced modest clinical responses in patients with
advanced/refractory HD. Treatment with monoclonal antibodies, however,
is limited by the
transience of their effects, and by poor bio-distribution in tumors
[12] . By contrast, T lymphocytes
redirected to eliminate CD30+ tumor cells through the expression
of a chimeric antigen receptor
(CAR) specifically binding the CD30 molecule [12,13]
have the potential to generate a sustained
anti-tumor effect.
Immunotherapy based on the transfer of redirected T cells is currently
under
investigation for a wide variety of tumors [14-18]
. Although preliminary clinical trials showed that
redirected T cells can function in vivo, they do not expand
or persist long-term [19,20] . Since
incomplete activation of redirected T cells after engagement of
the CAR contributes to the
limited activity and persistence of redirected T cells, we and others
have proposed grafting the
CAR on antigen-specific T cells [16, 21]
in order to provide them with co-stimulation from antigen
presenting cells when their native T-cell receptor (TcR)
is engaged. We chose EBV-specific
CTLs as the vehicle for CARs, as most individuals are persistently
infected with EBV, and
express viral antigens in epithelial cells and B lymphocytes [22]
. Hence, redirected T cells should
receive appropriate co-stimulation for long-term persistence when
they engage EBV-infected B
cells with their native TcR, thereby increasing their anti-tumor
activity mediated by engagement
of their chimeric receptor. This strategy holds particular appeal
for the treatment of EBV+ HD,
since the administration of CAR+ CTLs with their native receptor
specific for EBV-antigens and
their chimeric receptor specificity for CD30 should greatly reduce
the risk of tumor escape due
to EBV antigen or genome loss.
In the present study we explore the feasibility and value of redirecting
EBV-CTLs to
target CD30+ tumor cells, including HD tumor cells, ex vivo
and in vivo. Since CD30 is also
expressed on a subpopulation of activated T cells, we investigated
whether these CD30-redirected
EBV-CTLs would destroy T cells responding to unrelated immune stimuli,
and would
thereby producing undesirable generalized immunosuppression.
MATERIALS AND METHODS
Tumor cell lines.
The HD-derived cell lines HDLM-2, L428, L540 and KM-H2 and the anaplastic
large cell
lymphoma (ALCL)-derived cell line Karpas-299 (all CD30+ and
EBV-) were obtained from the
German Collection of Cell Cultures (DMSZ, Braunschweig, Germany).
Daudi, BJAB, Raji, HSB-2
and K562 were obtained from the American Type Culture Collection
(ATCC, Rockville, MD,
USA). The SP-53 cell line was kindly provided by Dr Amin Hesham
(MD Anderson Cancer
Center). EBV-infected B cells lines were generated as previously
described 23,24 . All cells were
maintained in culture with RPMI-1640 medium (Hyclone, Logan, Utah)
containing 10% fetal
bovine serum (FBS, Hyclone), 2 mM L-glutamine (GIBCO-BRL, Gaithersburg,
MD). Cells were
maintained in a humidified atmosphere containing 5% CO2 at 37°C.
Retroviral constructs.
CD30zeta chimeric T cell receptor (CD30CAR).
As previously described [13] , the CD30-specific
single chain Fv fragment was cloned in frame with the sequence encoding
the human IgG1
CH2-CH3 domains and the transmembrane and cytoplasmic domain of
the TcR receptor zeta
chain. This construct was then subcloned into the SFG retroviral
backbone [15] . To produce the
retroviral supernatant, 293T cells were co-transfected with Peg-Pam-e
plasmid containing the
sequence for MoMLV gag-pol, and the DRF plasmid containing the sequence
for the RD114
envelope [25] , using the Fugene6 transfection
reagent (Roche, Indianapolis, IN), according to the
manufacturer’s instructions [15, 26]
. Supernatant containing the retrovirus was collected 48 and 72
hours later.
Luciferase vectors.
The generation of retrovirus vectors encoding Firefly Luciferase
gene
(FFLuc) or the fusion protein eGFP-Firefly Luciferase (eGFP-FFLuc)
has been previously
described [15] . The FFluc specific vector, which
also contains the puromycin resistance gene, was
used for stable transduction of tumor cell lines. To confirm transgene
expression, 5x106 tumor
cells were lysed, aliquots diluted in 100 ul of D-luciferine according
to the manufacturer’s
instructions (Promega, Madison, WI). Bioluminescence was measured
in a luminometer
(Monolight, BD Biosciences Pharmingen, San Diego, CA). The eGFP-FFLuc
vector was used to
transduce the EBV-CTLs. GFP expression by transduced cells was evaluated
by FACS
analysis, while expression of FFLuc was detected as described above.
Preparation of CD30CAR EBV-CTLs.
Generation and transduction of EBV-CTLs with CAR.
EBV-CTLs were generated from 8 EBV-seropositive healthy donors and
from 4 patients with HD, as previously described [27]
. Established CTL lines, obtained after 3 stimulations with the autologous
EBV-infected
lymphoblastoid cell lines (LCLs), were transduced with retroviral
supernatant. For transduction,
5x105 EBV-CTLs were plated per well in a retronectin
(FN CH-296; Takara, Otsu, Japan)-coated
24-well plate in 0.5 mL of complete media (RPMI1640 [Hyclone] 45%,
Click’s medium [Irvine
Scientific, Santa Ana, CA] 45%, supplemented with 10% FBS and L-glutamine)
containing
recombinant human interleukin-2 (rhIL-2, 100U/mL; Proleukine; Chiron,
Emeryville, CA). Two ml
of CD30CAR retroviral supernatant were added to each well. As negative
controls, we used
non-transduced (NT) EBV-CTLs and EBV-CTLs transduced with
an irrelevant chimeric receptor
(14g2a zetaCAR) directed against the “GD2” antigen expressed
on neuroblastoma cells, but absent
on HD cells and EBV+ target cells [16] . These
14g2a
zetaCAR+ EBV-CTLs are referred as control
CTLs throughout the manuscript. Three days post transduction, cells
were removed from the
retronectin plates and expanded by weekly restimulation with LCLs
in the presence of rhIL-2
(50-80 U/ml).
Phenotype.
The following MAbs conjugated with phycoerythrin (PE), fluorescein
isothiocyanate
(FITC) and/or periodin chlorophyll protein (PerCP)
were used: CD3, CD4, CD8, CD19, CD16,
CD56, CD28 and CD30 (all from BD Bioscience). Expression of the
CAR on EBV-CTLs was
detected using Cy-5-conjugated goat anti-human IgG (H+L)
Abs (Jackson ImmunoResearch
Laboratories, West Grove, PA) which recognizes the human IgG1-CH2CH3
component
incorporated within the CAR. Cells were analyzed by a FACSCAlibur
(BD Biosciences)
equipped with the filter set for four fluorescence signals. The
antigen specificity of the CTLs
native receptors was evaluated with EBV-specific tetramers, as previously
described [27] . We
chose tetramers based on donor HLA type, which recognized: a) EBV-peptides:
EBNA3A, HLA-B8:
QAKWRLQTL, HLA-B7: RPPIFIRLL, LMP2, HLA-A2: CLGGLLTMV; BZLF1, HLA-B8:
RAKFKQLL (listed in Khanna and Burrows [28] ;
Houssaint et al. [29] ); b) CMV-peptides: HLA-A2:
NLVPMVATV and HLA-B7: TPRVTGGGAM [30] ; c) adenoviral-peptides:
HLA-A1: TDLGQNLLY;
HLA-A24: TYFSLNNKF; HLA-B7: KPYSGTAYNSL [31]
. Tetramers were prepared by the Baylor
College of Medicine core facility. For each sample, a minimum of
100,000 cells were analyzed
using a FACSCalibur with the CellQuest software (BD Biosciences).
TcR Vb usage by EBV-CTLs was studied using
the TcR Vb repertoire kit (IOTest Beta Mark
kit,
Immunotech, France) according to the manufacturer’s instructions.
Evaluation of cytotoxic activity.
To determine whether CD30CAR+ EBV-CTLs were able to
maintain specificity for autologous LCLs and kill EBV-/CD30+ targets,
we used a standard 51Cr
release assay [27] . As EBV-/CD30+ tumor cell
lines we used the HD cell lines HDLM-2, L428,
L540 and KM-H2, and the ALCL cell line Karpas-299 (see Supplemental
Table 1). As CD30
negative target cells we used Raji, Daudi, BJAB or SP-53 tumor cells
(all <0.1% CD30+).
Autologous phytohemoagglutinin (PHA)-stimulated peripheral
blood mononuclear cells (PBMCs)
were used as target cells to exclude autoreactivity, and were generated
by adding 5ug/ml of
PHA (Sigma, St Luis, MO) to PBMCs on day 0 and then rhIL-2 (100U/ml)
on day 3 and 6 (see
Supplemental Table 1). In
some experiments, to evaluate susceptibility of activated T cells to
CAR+ CTLs mediated killing, autologous T blasts were transduced
with a retroviral vector to
stably over-express the CD30 molecule. In some experiments, before
labeling, target LCLs
were depleted or enriched for CD30 expression using immuno-magnetic
beads (MACS,
Miltenyi, Auburn, CA). To determine whether killing was restricted
by HLA class-I or class-II
MHC molecules or was dependent on CD30, target cells were preincubated
for 30 minutes
either with the MAb W6/32 (Dako, Carpinteria, CA) that recognizes
a monomorphic HLA class-I
determinant or the monoclonal antibody CR3/43 (Dako), recognizing
HLA-DR, DP and DQ, or
with the MAb BerH2 (Dako), that recognizes the CD30 molecule. Killing
by CAR+ CTLs was
also evaluated in the presence of serum collected from patients
with advanced HD, and tested
for the presence of soluble CD30 using a specific ELISA kit (Alexis,
Axxora, San Diego, CA).
Serum samples with >80U/ml of soluble CD30 were used.
Co-culture experiments.
To evaluate the ability of CD30CAR+ CTLs to completely eliminate
CD30+ tumors we co-cultured 1x106 EBV-CTLs in 24-well
plates, in the presence of non
irradiated CD30+ tumor cells (HDLM-2, L428, L540 or Karpas-299,
at 4:1 E:T ratio), all in the
presence of rhIL-2 (50 U/ml). Parallel co-cultures were plated with
control CTLs. Phenotypic
analyses were performed on days 5 and 8. T cells were detected using
CD3-PerCP, LCLs with
CD19-PE and tumor cells with CD30-FITC MAbs (BD Biosciences).
Elispot.
The following EBV peptides were used for analysis of EBV-specific
T-cell populations
according to the donors' HLA specificity: EBNA3A, HLA-B8: QAKWRLQTL,
FLRGRAYGL; HLA-B7:
RPPIFIRLL, LMP2, HLA-A2: CLGGLLTMV; BZLF1, HLA-B8: RAKFKQLL; BMLF1,
HLA-A2:
GLCTLVAML (listed in Khanna and Burrows [28]
; Houssaint et al. [29] ). For some experiments the
CMV-peptides A2-NLV and B7-TPR were used. Peptides were synthesized
by Genemed
Synthesis (South San Francisco, CA). In this paper, the peptides
are referred to by the first 3
amino acids as underlined. To evaluate the functionality of CAR+
EBV-CTLs, we measured
reactivity to identical EBV-derived peptide epitopes using the Interferon-g
Elispot assay [27] .
Briefly, CTLs were plated in triplicate and serially diluted from
5x104 to 5x102 cells/well, and then
100 µL of appropriately diluted peptides (5 uM) or autologous,
irradiated LCLs (1×105 cells),
were added to the wells as positive controls. Negative controls
included CTLs alone and CTLs
loaded with irrelevant peptides.
Proliferation of CAR+ CTLs.
Control and CAR+ CTLs were labeled with 1.5 uM of
carboxyfluoroscein succinimidyl ester (CFSE, Invitrogen,
Carlsbad, CA) following the
manufacturer’s instruction, and then plated at 1x106
EBV-CTLs in 24-well plates, in the
presence of irradiated autologous EBV+ or CD30+ cells (at 4:1 E:T
ratio), and with rhIL-2 (50
U/ml). On day 5, cells were labeled with CD8 PerCP and EBV+ tetramers-PE.
Samples were
analyzed by FACS and cell division assessed by CSFE dilution.
Generation of Cytomegalovirus- (CMV) or Adenovirus (Ad)-specific T cells.
Ad-specific T cells were generated from 3 Ad-seropositive healthy
donors, as previously
reported [31] . Briefly, PBMCs rested overnight
in serum-free media, were infected with Ad5f35
adenoviral vector for 2 hours at an MOI of 200 at 37 oC
and then plated at 2x106 /well in a 24-well
plate. Similarly, CMV-specific T cells were reactivated from 4 CMV-positive
healthy donors, by
infecting PBMCs with Ad5f35-pp65 vector at an MOI of 50, for 2 hours
at 37 oC, and then plating
the cells at 2x106 /well in a 24 well plate. Autologous
non-transduced, control CAR or
CD30CAR+ EBV-CTLs were added to these cultures at a 5:1 (PBMCs:
CTL) ratio. The
percentage of tetramer+ T cells binding to the hexon-tetramer or
to the pp65-tetramer were
evaluated by FACS analysis after 8-9 days of co-culture. Using this
method, adeno-specific or
CMV-specific T cells could be reactivated in all the tested donors.
The presence of transduced
CTLs was demonstrated in the co-cultures by using anti-human IgG
(H+L) Ab. To confirm the
functionality of the reactivated T cells, the frequency of IFN-g
producing
cells was determined
using the IFN-g Elispot assay against
CMV- or Adenoviral-derived peptides.
In vivo experiments.
To assess the homing, persistence and anti-tumor effects of CD30CAR+
EBV-CTLs in vivo, we
used a SCID mouse model and the IVS imaging system (Xenogen).
Transduction of tumor cells and EBV-CTLs with luciferase vectors.
The CD30+ tumor cell line
L428 was transduced with the FFLuc vector on retronectin-coated
plate, and then selected in
puromycin (Sigma). CAR+ EBV-CTLs were first enriched to ensure CAR
expression on >90% of
EBV-CTLs and then transduced on a retronectin-coated plate with
the eGFP–FFluc vector.
Phenotypic analysis confirmed that the GFP was expressed on CAR+
CTLs.
Mice.
Six to eight week old CB17/SCID mice were purchased from Harlan-Sprague
(Indianapolis, Indiana). All mouse experiments were performed in
accordance with Baylor
College of Medicine Animal Husbandry and IACUC guidelines.
Efficacy of CD30CAR+ CTLs against EBV+ tumors. To evaluate whether
CAR+ EBV-CTLs
retained their anti-tumor activity against the native EBV+ tumor
(LCLs), we used SCID mice that
were sublethally irradiated (230 cGy) and injected s.c. the following
day with 1x107 autologous
LCLs resuspended in matrigel [32] . A third group
of mice was implanted s.c. with HLA-mismatched
LCLs. Ten-15 days later, when tumor was measurable (0.5-0.8 cm in
max diameter), mice
received 1x107 eGFP-FFluc labeled CD30CAR+ EBV-CTLs or
control CTLs i.v.. After CTL
transfer, mice received i.p. twice weekly 500-1000U of rhIL-2. CTLs
trafficking towards EBV+
tumors and their expansion at the tumor site were measured using
the bioluminescence
approach described above. Thirty days after CTL transfer, mice were
euthanized and evaluated
for presence of EBV+ tumor.
To confirm that increased bioluminescence signals corresponded to
increased T-cell numbers
rather than exclusively to increased transgene expression in a fixed
number of cells, we
euthanized mice showing increasing levels of bioluminescence. Cells
from the tumor site were
isolated and stained with anti-human CD45 and CD3 antibodies.
Anti-tumor activity against CD30+ HD tumors.
We used an intraperitoneal (i.p.) tumor xenograft
model to test the efficacy of CAR+ CTLs against EBV-/CD30+ HD [21]
and to evaluate the effects
of concomitant stimulation of the native-TcR receptor on these CD30CAR+
EBV-CTLs using
EBV-infected cells (LCLs). To assess the anti-tumor activity
of CAR+ CTLs, CD30+ tumor cells
labeled with FFLuc gene were injected intraperitoneally (5x106
cells) in sublethally irradiated
(230 cGy) SCID mice. Five to 7 days later, the mice received two
injections 4 days apart of
either control or CD30CAR+ EBV-CTLs (1x107 ). rhIL-2
(500-1000U) was administered i.p every
other day. To test the effects of native TcR stimulation, a third
group of mice received i.p.
injections of irradiated (40Gy) autologous LCLs (4x106
) twice/week for 2 weeks. Tumor growth
was monitored twice a week by injecting mice i.p. with D-luciferin
(150 mg/kg). Photon emission
was analyzed using the Xenogen-IVIS Imaging System. Briefly, a constant
region-of-interest
(ROI) was drawn over the tumor region and the intensity of
the signal measured as total
photon/sec/cm2 /sr (p/s/cm2 /sr) as
previously validated [15] .
Statistical analysis.
All in vitro data are presented as mean ± SD. Student’s
t test was used to determine the
statistical significance of differences between samples, and P
<
.05 was accepted as indicating
a significant difference. For the bioluminescent experiments, intensity
signals were log-transformed
and summarized using mean ± SD at baseline and multiple subsequent
time points
for each group of mice. Changes in intensity of signal from baseline
at each time point were
calculated and compared using paired t-tests or Wilcoxon signed-ranks
test.
RESULTS
EBV-CTLs can express the CD30CAR whilst retaining their phenotype and native receptor function.
The transduction efficiency of CD30CAR was 26% +/- 11% (Fig.
1A) in 8 different EBV-CTL lines from healthy donors.
Figure 1. Growth kinetics, immunophenotype and functionality
of EBV-CTL lines are
retained after transduction with CD30CAR.
Figure 1. Growth kinetics, immunophenotype and functionality of
EBV-CTL lines are
retained after transduction with CD30CAR.
EBV-CTL lines were expanded from PBMCs obtained from 8 healthy EBV-seropositive donors by weekly stimulation with irradiated autologous LCLs and bi-weekly feeding with rhIL-2. EBV-CTLs were transduced with the CD30CAR or an irrelevant CAR (control CTLs) after the 3rd stimulation.
Fig. 1A shows CD30CAR expression evaluated by flow
cytometry using a goat anti-human IgG (H+L) Ab (solid
line) or the isotype control (dotted line).
Fig. 1B shows the growth of EBV-CTLs transduced with the CD30CAR (open square) or an irrelevant CAR (solid square). The arrow indicates time of retroviral transduction.
Fig. 1C shows the expression of CAR on transgenic CTLs over time. The number of stimulations after transduction are indicated. Bars represent average +/- SD for the 8 donors.
Fig. 1D shows the immunophenotype of the EBV-CTLs transduced with the CD30CAR (solid bars) compared to EBV-CTLs transduced with an irrelevant CAR (white bars). Gray bars show the phenotype of the CD30CAR+ CTL stimulated with CD30+ tumor cells. Means +/- SD are shown for the 8 donors.
Fig. E shows that CD30CAR can be detected on both CD8+ (left plot) and CD4+ (right plot) transduced EBV-CTLs.
After transduction, EBV-CTLs were maintained in
culture by weekly restimulation with autologous LCLs in the presence
of rhIL-2. To ensure that
CD30CAR transduced CTLs retained the same phenotypic and functional
characteristics as the
control EBV-CTLs, we monitored their growth kinetics and immunophenotype
for a minimum of
4 weeks. Expansion of CD30CAR+ CTLs was comparable to that of control
CTLs (Fig. 1B). In
addition, expression of the CD30CAR was retained for the entire
culture period (Fig. 1C). The
CTLs ceased to proliferate and progressively died when re-stimulation
with LCLs and rhIL-2
were stopped, confirming that the presence of the CD30CAR did not
alter the requirement for
stimulation with antigen and exogenous cytokine, and demonstrating
that autonomous growth
had not developed (data not shown).
Transduction with CD30CAR did not significantly change the immunophenotype
of the EBV-CTLs
(CD3+/CD8+: 84% +/- 11%; CD3+/CD4+: 11% +/- 8%). The majority
of control EBV-CTLs
were CD3+/CD8+ T cells (84% +/- 12%), with 12% +/- 11% of CD3+/CD4+
T cells. Less than 2% of
the CTLs were CD3-/CD56+/CD16+ (Fig. 1D). The
costimulatory molecule CD28 was
expressed on 86% +/- 15% of control CTLs and 78% +/- 23% of CD30CAR+
CTLs. The
immunophenotype of transgenic CTLs as well as the expression of
CD30CAR remained
unchanged after stimulation with CD30+ HD tumor cells (see Table
2 and Fig. 1D). Expression
of CD30CAR was detected on both CD8 and CD4 CTLs (Fig.
1E).
EBV-CTLs expressing the CD30CAR kill CD30+ target cells in an MHC unrestricted manner.
To discover whether transgenic expression of CD30CAR on EBV-CTLs
would render
them capable of killing CD30+ tumor cells, we used both a standard
5 hour 51Cr release assay
and a long-term co-culture assay.
Cytotoxicity assay.
CTLs expressing CD30CAR lyzed the MHC mismatched CD30+ HD cell line
HDLM-2 at a significantly higher rate (50% +/- 20% at 20:1 E:T ratio) compared
to control CTLs (12% +/- 11%) (p<0.05) (Fig. 2A).
Figure 2. CD30CAR transduced EBV-CTLs specifically lyse CD30+
targets.
Figure 2. CD30CAR transduced EBV-CTLs specifically lyse CD30+ targets.
Fig. 2A shows the results of a standard 51Cr
release assay of several CD30+ tumor cell lines, at a CTL:tumor
cell ratio of 20:1. Bars represent the mean ± SD of the EBV-CTLs
generated from 8 donors and transduced with the CD30CAR (black bars)
or an irrelevant CAR (white bars) (p<0.05).
Fig. 2B shows that killing (shown is the % of lysis
at 20:1 E:T ratio) of the CD30+ targets (black bar) by
CD30CAR is inhibited by incubation with CD30 MAb (striped bar)
but not by isotype control MAb
(white bar) or by class-I MHC MAb (gray bar), indicating
that killing of CD30CAR is not MHC
restricted (p<0.05).
Fig. 2C shows that EBV-CTLs expressing the CD30CAR
can eliminate CD30+ tumor cells in a long-term culture assay. EBV-CTLs
obtained from healthy donors and transduced either with irrelevant CAR
(left panels) or CD30CAR (right panels) were co-cultured
with the indicated CD30+ tumor cell lines (ratio 5:1). After 5-7 days of
culture, cells were collected and stained with CD3-PerCP and CD30-FITC
to evaluate the growth of CD30+ tumor cells. No CD30+ cells were
detectable after co-culture with the CD30CAR+ EBV-CTLs, while CD30+ cells
were detectable when tumor cells were co-cultured with control CTLs. The
phenotypes shown are representative of 4 performed experiments.
To exclude acquisition of autoreacivity by transduced CTLs, we tested
their cytotoxicity against
autologous PHA-activated blasts. These cells express negligible
amounts of CD30 (4.5 +/- 2%) by
day 8 when they were used as target cells (Supplemental
Table 2). As expected, no significant
lysis of autologous PHA blasts (<10%, at 20:1 E:T ratio) was
produced by non-transduced or
CAR+ EBV-CTLs (Fig. 2A and Supplemental
Fig. 2). Both HSB-2 and K562 express the CD30
molecule, so a contribution from natural killer (NK) activity
to the killing of these cells could not
be ruled out, although NK cells represented less than 2% of the
population, as assessed by
immunophenotype (CD3- CD56/16+ were <2%).
To determine if killing mediated by CD30CAR was impaired by the presence
of soluble CD30,
we repeated the cytotoxicity assay in the presence of serum obtained
from patients with
advanced HD, in which we measured high levels of soluble CD30 molecule
(>80 U/ml). Using a
1:1 ratio of serum:medium, killing of CD30+ targets by redirected
CTLs was not significantly
affected (lysis of HDLM-2 at 20:1 E:T ratio 74 +/- 9% w/o
serum vs 61 +/- 8% with serum, data not
shown). This result was expected as the single chain for this CD30
molecule was previously
reported not to be inhibited by the specific soluble molecule [13]
.
Co-culture experiments.
To evaluate the long-term ability of EBV-CTLs expressing the
CD30CAR to eliminate CD30+ tumor cells, we co-cultured 1x106
EBV-CTLs with non irradiated
CD30+ tumor cells (HDLM-2 or L428 or Karpas-299), in the presence
of rhIL-2 (50 U/ml).
Parallel co-cultures were plated with control CTLs. Cells were collected
after 5-8 days of culture
and tumor cells enumerated by CD30 staining and FACS analysis. In
the presence of
CD30CAR+ CTLs we found complete elimination of CD30+ tumor (CD30+
cells <1%), while
tumor cells overgrew (CD30+ cells: 30-45%) in cultures with control
CTLs (Fig. 2C).
EBV-CTLs continue to kill EBV-expressing targets through their native receptor.
We also used the standard 5 hour 51Cr release assay
to confirm that transgenic expression of CD30CAR
on EBV-CTLs did not reduce their ability to recognize the targets
of their native receptors (i.e.
autologous EBV+ LCLs). As shown in Fig. 3A, CTLs
expressing CD30CAR, while able to kill
CD30+ allogeneic tumor cells, retained their cytotoxic activity
against autologous LCLs
(65% +/- 14%, at 20:1 E:T ratio).
Figure 3. EBV-CTLs expressing the CD30CAR retain their ability
to kill EBV+ tumor cells.
Figure 3. EBV-CTLs expressing the CD30CAR retain their ability to kill EBV+ tumor cells.
Fig. 3A shows the mean (+/- SD) 51Cr release from target cells exposed to EBV-CTLs from 8 donors transduced with an irrelevant CAR (on the left) and CD30CAR (on the right). CD30CAR+ CTLs lysed autologous LCL (open squares) and the EBV-/CD30+ HD derived cell line HDLM-2 (filled circles) (p<0.05), while control CTLs showed significant lysis only of autologous LCL. Auto-reactivity was excluded by the absence of lysis of autologous PHA blasts (asterisk).
Fig. 3B shows that CD30CAR+ CTLs (black bars)
retain their killing activity against autologous LCLs
and acquire the ability to kill allogeneic LCLs (p<0.05).
As LCLs express CD30, this suggests that the observed lysis of allogeneic
LCLs is mediated by the CAR.
Fig. 3C shows that killing (at 20:1 E:T ratio)
by CD30CAR+ CTLs of autologous LCLs (black bar) is not inhibited
by incubation with class-I MHC MAb (gray bar). This suggests that
that killing of LCLs can still be
mediated by the engagement of the CAR with the CD30 molecule expressed
on LCLs.
Fig. 3D shows the expression of CD30 antigen on LCLs after depletion or enrichment for the CD30 molecule using immunomagnetic beads (MACS system) in one representative donor. The percentage of specific 51Cr release at 40:1 E:T ratio of CTLs against CD30 depleted and CD30 selected autologous LCLs in 3 donors is shown in the bottom panel.
To determine whether CD30CAR+ CTLs retained full activity mediated
through their native
receptor, we quantified EBV-mediated killing by labeling LCL after
depletion of CD30+ cells,
using the MACS system. LCLs-depleted for CD30 were then used as
target cells in these 51Cr
release assays (Fig. 3D). We observed that lysis
of CD30 negative LCL by CAR+ CTLs was
comparable to that of control EBV-CTLs, suggesting that recognition
and lysis of EBV-infected
cells was retained after transduction (Fig. 3D).
The generation of antigen-specific T cells is not impaired by the presence of EBV-CTLs expressing the CD30CAR.
As CD30 is expressed by a subpopulation of activated T cells after
exposure to viral antigens and mitogens [33]
, it is possible that CD30CAR+ CTLs would delete viral
or other antigen-specific T cells during exposure to an infectious
agent, with deleterious
consequences for immunity. We therefore determined if cultured CD30CAR+
CTLs would
display a reduced EBV-antigenic repertoire (self-destruction),
or if they could inhibit the ability of
co-cultured primary T cells to respond to other antigenic stimuli,
such as CMV and adenovirus
(bystander destruction). We first measured CD30 upregulation
on T cells during our culture
conditions. We collected PBMCs from 8 healthy EBV-seropositive donors
and activated them
with irradiated autologous LCLs. Expression of CD30 on CD3+ T cells
was then evaluated daily
by flow cytometry. CD30 was not detectable on resting T cells (<1%),
but, was transiently
upregulated on a subpopulation of CD3+ T cells (14% +/- 8%) between
day 5 and 7 after the first
stimulation with LCLs (Table 1).
Antigenic repertoire of EBV-CTLs.
Since weekly restimulation with LCLs is used to expand
transduced CTLs, and since this antigen-specific stimulation can
induce upregulation of CD30,
we investigated the effects of CD30CAR expression by EBV-CTLs on
their own EBV-specific
TcR repertoire. We evaluated the frequency of CTLs responding to
available HLA class-I
restricted EBV-peptides using specific tetramers before and after
expression of CD30CAR. The
antigenic repertoire of the EBV-CTLs did not change after
transduction in any of the 5 donors
tested. Instead the percent of tetramer+ CTLs recognizing lytic,
immunodominant and less
immunogenic latent EBV-antigens was comparable in CTLs expressing
CD30CAR and CTLs
expressing an irrelevant CAR (Fig. 4A).
Figure 4. EBV-CTLs redirected with CD30CAR retain their polyclonal
EBV specificity.
Figure 4. EBV-CTLs redirected with CD30CAR retain their polyclonal EBV specificity.
Fig. 4A shows the frequencies of tetramers recognizing
lytic (BZLF1-RAK) or latent (EBNA3C-RPP
and LMP2-CLG) EBV-associated antigens in control and transgenic
EBV-CTLs generated from
3 different donors. The lower panels show that the same frequency
of EBV-specific tetramers is
maintained after transduction with CD30CAR.
Fig. 4B shows that the CD30CAR is also detectable on tetramer+ CTLs.
Fig. 4C shows CFSE labeled control (left panels) and CD30CAR+ CTLs (right panels) non stimulated (upper panels) stimulated with EBV+ (middle panels) or CD30+ cells (lower panels). After LCLs stimulation (middle panels) both control and CD30CAR+ EBV-tetramer+ CTLs proliferate, as shown by decrease of CFSE+ cells. After stimulation with CD30+ cells (lower panels) only CAR+ CTLs proliferate.
Fig. 4D shows the frequency of T cells responding
to EBV-specific peptides in control and CD30CAR+ EBV-CTLs from a representative
donor, assessed by IFN-g Elispot assay. Tetramer
and Elispot analyses
are representative of a total of 5 donors.
To determine whether there were changes in CTL clonality following
transduction, we analyzed
the TcR-combinatorial diversity of control and EBV-CTLs expressing
CD30CAR by monoclonal
antibodies recognizing TcR-Vb specific
regions. Redirected CTLs had a TcR-Vb repertoire
superimposable on that of non-transduced EBV-CTLs, with maintained
heterogeneity and no
specific over- or under-representation of any Vb
family
(data not shown).
Reactivation of CMV- or Ad-specific-T-cell responses is not affected.
We also determined if CD30CAR grafted CTLs could affect the reactivation
of bystander antigen-specific T-cell responses. We added CD30CAR+ EBV-CTLs
to PBMCs, which were then stimulated to
reactivate CMV- or Ad-specific CTLs. Virus-specific CTLs were successfully
reactivated in all
donors (Fig. 5A and Table 3),
even though CD30CAR+ CTLs persisted to the end of the co-culture
experiments (Fig. 5B).
Figure 5. Reactivation of viral-specific T cells is not impaired
in the presence of EBV-CTLs
expressing the CD30CAR.
Figure 5. Reactivation of viral-specific T cells is not impaired
in the presence of EBV-CTLs
expressing the CD30CAR.
Autologous EBV-CTLs engineered to express the CD30CAR
were added to cultures of PBMCs stimulated to reactivate CMV- or
adenovirus-specific CTLs.
Fig. 5A shows the percent of pp65-tetramer+ T cells
generated in a representative donor by day
9 of culture in the presence of non transduced EBV-CTLs (upper
plot), EBV-CTLs transduced
with an irrelevant CAR (middle plot) or the CD30CAR+(lower
plot).
Fig. 5B: cells from the co-culture
were stained with the goat anti-human IgG (H+L) Ab to demonstrate
the continued
presence of the CD30CAR+ CTLs throughout the culture. As expected,
no CAR+ CTLs were
detectable in co-cultures where NT CTLs were added. In contrast,
21% and 25% CAR+ CTLs
were detectable at the end of the co-cultures where irrelevant-CAR
or CD30CAR+ CTLs were
added, respectively.
Fig. 5C shows the IFN-b specific Elispot assay of co-culture from 2 representative donors. Mean frequency (?SD) of IFN-b producing T cells in response to the CMV-specific peptides NLV and TRP is shown.
Fig. 5D shows the percent of Adeno-tetramer+ T cells (shown is the analysis with the two available tetramers) in the only donor whose viral specific response was reduced when CD30CAR+ EBV-CTLs were added to the culture (see also Table 3).
EBV-CTLs generated from patients with HD can be grafted with a functional CD30CAR.
To ensure that the approach we describe would also be feasible using
T cells from patients with
active HD, we generated EBV-CTLs from 4 patients with relapsed HD
and transduced them with
the CD30CAR. As observed for healthy donors, EBV-CTLs were efficiently
grafted with the
CD30CAR, as 22% +/- 5% of EBV-CTLs stained with the anti-human IgG
(H+L) Ab (Fig. 6A).
Figure 6. EBV-CTLs generated from patients with HD can be grafted
with a functional
CD30CAR
Figure 6. EBV-CTLs generated from patients with HD can be grafted
with a functional
CD30CAR. EBV-CTL lines were expanded from PBMCs
of four patients with HD.
Fig. 6A shows the expression of CD30CAR on two
representative CTL lines by flow cytometry using a goat
anti-human IgG (H+L) Ab (solid line). The dotted line
shows the isotype control.
Fig. 6B shows the immunophenotype of EBV-CTLs generated
from these 4 HD patients and transduced with
the CD30CAR (black bars) compared to EBV-CTLs transduced
with an irrelevant CAR (white
bars). Mean and SD are shown.
Fig. 6C shows the frequency of tetramers recognizing the lytic (BZLF1-RAK) EBV-associated antigen in EBV-CTLs generated from one of these patients. The lower panels show that the same frequency of EBV-specific tetramers is maintained after transduction with CD30CAR and that the CD30CAR is also detectable on tetramer+ T cells.
Fig. 6D shows the killing of LCLs and CD30+ tumor
cell lines, in a standard 51Cr release assay at a
CTL:tumor cell ratio of 20:1. Bars represent the mean ±
SD of the EBV-CTLs transduced with
the CD30CAR (black bars) or an irrelevant CAR (white bars).
CD30CAR+ CTLs lysed both
autologous LCLs and CD30+ target cells, while control CTLs showed
significant lysis only of
autologous LCLs.
Redirected EBV-CTLs home to native EBV+ tumors in vivo and subsequently expand.
To demonstrate CD30CAR+ EBV-CTLs maintain their functional activity
in
vivo, we used a
mouse xenograft model [32] . First, CTLs expressing
the CD30CAR were FACS sorted to obtain a
population of >90% CAR+ cells (Fig. 7A).
Figure 7. CD30CAR+ EBV-CTLs can control tumor growth in vivo,
while retaining their
ability to migrate to EBV+ tumor and expand.
Figure 7. CD30CAR+ EBV-CTLs can control tumor growth in vivo,
while retaining their
ability to migrate to EBV+ tumor and expand. To evaluate
in
vivo homing NT EBV-CTLs or
CTLs transduced with CD30CAR and sorted for transgene expression
were injected i.v. in SCID
mice implanted s.c. with autologous LCLs 32 . Both NT and CAR+ EBV-CTLs
were labeled with
the eGFP-FFLuc gene to monitor their trafficking and expansion,
using an in vivo imaging
system (Xenogen-IVIS Imaging System).
Fig. 7A shows that circa 50% of CD30CAR+ EBV-CTLs, as assessed by the goat anti-human IgG (H+L) Ab, are expressing the FFLuc transgene as GFP+.
Fig. 7B illustrates that the signal of EBV-CTLs localized to the EBV+ tumors and increased in mice receiving either control (upper panels) or CD30CAR+ EBV-CTLs (lower panels).
Fig. 7C shows the bioluminescence fold expansion of CTL at the tumor site. To evaluate the contribution of co-stimulation 21 by EBV-antigen, EBV-CTLs transduced with irrelevant CAR or CD30CAR were injected i.p. in SCID mice bearing EBV-/CD30+ L428 tumor that was transgenic for FFLuc. EBV-CTLs were transferred 7 days after tumor implant. Tumor growth was monitored using the in vivo imaging system.
Fig. 7D illustrates that by 7 days after CTLs infusion,
tumor growth measured as maximum photon/sec/cm 2 /sr (p/s/cm 2 /sr), was
significantly greater in mice receiving control CTLs (upper panels)
compared to mice receiving CD30CAR+ EBV-CTLs (middle panels). Persistence
of tumor control can be observed in mice
receiving CD30CAR+ EBV-CTLs and i.p injection of irradiated EBV-infected
cells, which thus provide the appropriate co-stimulation (lower panels).
Fig. 7E illustrates the results of 6 mice per group
implanted with the CD30+ L428 cell line. Bars represent average
of light emission +/- SD (p<0.05).
CD30CAR expressing EBV-CTLs have anti-tumor activity in vivo against EBV-/CD30+ tumors.
To assess whether CD30CAR+ EBV-CTLs can modify the growth of an established
EBV-/CD30+ tumor and to evaluate the co-stimulatory effects of EBV-infected
cells on the anti-tumor
activity of CAR+ CTLs, we used an i.p. SCID xenograft model [21]
. Sublethally irradiated
SCID mice were implanted with FFluc labeled CD30+ tumor cells (L428)
in the peritoneum.
Light emission was monitored as an indication of tumor growth. Once
progressive increase of
bioluminescence occurred (usually 7 days after tumor injection),
mice received control EBV-CTLs
or CD30CAR+ EBV-CTLs i.p. followed by rhIL-2 i.p. on alternate days.
To evaluate
costimulation from EBV-infected cells, a group of mice also received
irradiated autologous
EBV+ LCLs i.p. twice weekly for two weeks. After adoptive transfer,
we observed a reduction of
light emission in mice treated with CD30CAR+ EBV-CTLs (Fig.
7D and E), indicating control of
tumor growth for more than 2 weeks. In addition, the control of
tumor growth was sustained for
up to 4 weeks in mice that also received co-stimulation from autologous
EBV-infected cells (Fig.
7D and E). In contrast, photon emissions, and
thus tumor size, increased in mice receiving
control EBV-CTLs, regardless of co-stimulation from autologous EBV-infected
cells (Fig. 7D and E).
DISCUSSION
Adoptive transfer of viral-antigen specific CTLs has had clinical
value in patients with
EBV+ HD [6,7] . To increase the activity of these
cells, and reduce the potential for tumor escape by
target antigen downregulation, we have modified these viral antigen
specific cells so that they
express a transgenic artificial receptor specific for the HD associated
antigen CD30 [8] . We have
shown that EBV-CTLs expressing this CD30 specific receptor retain
their ability to kill EBV+
tumors and acquire the ability to recognize and kill CD30+ HD tumor
cells in vitro and in a SCID
mouse model in vivo. The redirected EBV-CTLs also retained
their phenotype and polyclonal
specificity for EBV-derived antigens. Moreover, T cells from HD
patients, which may have low
endogenous level of the zeta chain [6] , were
also able to produce EBV-CTLs that were able to
express functional levels of CD30CAR, confirming the feasibility
of our approach for patients
with this disease. When injected in vivo, the redirected
EBV-CTLs retained the ability to migrate
to EBV+ tumors, to expand locally in response to EBV-antigens through
their native TcR, and to
kill CD30+ tumor cells through their chimeric receptor, suggesting
they will receive sufficient and
appropriate co-stimulation from persistently infected normal and
malignant host cells to be able
to expand and survive long-term in humans with HD.
Although CAR targeting CD30 and other antigens can readily be expressed
in primary T
cells, which will then kill the relevant tumor targets in vitro
[13] , it has become evident from clinical
trials that CAR-expressing primary T cells have limited persistence
in
vivo after adoptive
transfer [19,20] . This lack of persistence has
been attributed to incomplete stimulation after
engagement of the CAR [19,20] , a phenomenon
particularly problematic when tumors that lack
expression of co-stimulatory molecules are targeted. To date, the
primary strategy to overcome
the limitations of these first generation CARs has been to incorporate
additional co-stimulatory
endodomains in the chimeric receptor [15, 35-38]
. In a range of studies, both in vitro and in vivo, the
addition of such endodomains improves CAR+ T-cell function, increasing
proliferation, cytokine
secretion and cytotoxic effector activity [15,
21,
35]
. While promising, this approach has the limitation
that it can never precisely mimic the physiological sequence of
co-stimulatory events that follow
engagement of the native antigen receptor. It is now clear that
optimum recruitment of an
immune response requires several discrete families of co-stimulatory
molecules to be employed
in sequence. Moreover, recruitment of each class of effector
cells (for example CD4 helper
versus CD8 cytotoxic) has a preferential requirement for a different
costimulatory receptor-ligand
interaction.
Because of the inherent limitations to chimeric receptor manipulation
described above, a
second strategy for improving efficacy has been investigated. Instead
of expressing the CAR in
an undefined population of primary T cells, it is possible to transduce
cultured cytotoxic T-lymphocyte
lines with specificity for antigens known to be expressed in
vivo on professional
APCs. Through an appropriate sequence of co-stimulation,
these APCs produce effective and
sustained immune responses. In this way, chimeric T cells receive
appropriate co-stimulatory
signals in correct sequence when they engage their native receptors,
and are thus better able to
persist and kill their tumor targets when they engage their chimeric
receptor. A number of pre-clinical
studies have suggested the benefit of this overall concept [16,
21]
. We chose to investigate
the value of expressing CD30CAR in EBV-CTLs because the lifelong
expression of EBV-antigens
in infected individuals ensures the long-term persistence of infused
CTLs, and because
EBV-CTLs have been shown to traffic to and destroy EBV-expressing
malignancies [23, 39] . We
reasoned that EBV-CTLs expressing the chimeric receptor would retain
these desirable
characteristics. Moreover, since the tumor cells from almost half
the patients with HD also
express EBV-antigens themselves, tumor cells could be targeted by
both native and chimeric
receptors on a single T cell, potentially enhancing effectiveness
and reducing the risk of a tumor
escape by antigen modulation/mutation [40] .
By using redirected EBV-CTLs, costimulation will be
provided locally by EBV-infected HRS cells in EBV+ HD tumors and
systemically by the EBV-infected
memory B cells that persist lifelong in lymphoid tissues.
We chose to target CD30 since this antigen is constitutively expressed
on virtually all
HRS cells both at diagnosis and at relapse [8]
. We used an antibody derived rather than a alpha-beta
TcR as the basis for the synthetic receptor to minimize issues of
competition for receptor
formation[41] , and to allow binding even to
malignant target cells in which the antigen processing
machinery had become impaired [40]. The CD30
antigen is not detected on cells of the peripheral
blood or on resting lymphocytes, but it is present on a subpopulation
of physiologically activated
T cells and on thymic medulla [33] . Expansion
of an anti-CD30-specific CTL population could
therefore be self-defeating, with auto-destruction as the CTLs became
activated, or the
destruction of T cells responding to other infectious antigens.
Our experiments demonstrated
that these hypothetical concerns were not realized in practice,
since CD30CAR+ EBV-CTLs do
not lose their own target antigen specificities, nor do they impede
reactivation of CMV- and
adenovirus-specific CTLs. Preservation of virus-specific CTL reactivation
may relate to the weak
expression of CD30 on activated T cells, which is likely insufficient
for killing by CD30CAR
effector cells. Alternatively, CD30 may be present on only a minor
subset of activated T cells,
which is non-essential to an effective immune response. This latter
explanation is favored by
studies in CD30 knockout mice, in which the number of circulating
B and T lymphocytes, and
the function of their immune system, is largely unimpaired [42]
. Although clinical trials will be
required to confirm the safety of this strategy, we believe that
discrimination between levels of
CD30 expression on physiologically activated T cells and on HRS
tumor cells will occur in vivo
and that activated T cells will be spared by the CD30CAR+ EBV-CTLs
that can kill tumor
targets. As far as expression of CD30 on thymic medulla is concerned,
impaired negative
selection of T cells has been described in CD30 knockout mice [42]
. However, this process is
minimal in adult individuals. Expression of CD30 on other non-hematopoietic
cells is limited to
malignancies, such as embryonal carcinomas and some mesenchymal
tumors [42] .
Our clinical trials of adoptive transfer of EBV-CTLs for therapy
of PTLD and EBV+ HD
demonstrated that infused EBV-CTLs localized to the tumor and expanded
[7, 23, 39] . Moreover,
although these cells lacked (central) memory markers CCR7 and CD62L
(data not shown) they
persisted for >8 years, and could be expanded by appropriate antigenic
stimuli, thereby
ensuring both early and long-term control of disease [6,7,
23,
39]
. While the pathway of differentiation
of human T cells into the memory compartment remains ill defined,
it seems likely that even
terminally differentiated effector cells can nonetheless revert
to effector memory [43] . This
observation appears to hold both for EBV-specific [7,
23,
39]
and melanoma antigen (MART-1)-specific
effector cells [43] . Our SCID mouse xenograft
model indicated that we can anticipate the
CAR+ CTLs will behave in a similar manner, since the phenotype of
these cells and their
homing ability to EBV+ cells was identical to that of non-transduced
EBV-CTLs. Prolonged T
cell culture may reduce the efficacy of T cell therapy [44]
, but our approach uses a polyclonal CTL
population that has been validated for its ability to expand, persist
and function in vivo [7, 23,
39]
.
CD30 is also expressed by EBV negative HRS cells, so that in principle
our approach
could be extended to all HD, regardless of the presence of EBV antigens.
However, EBV
negative HD, unlike EBV+ disease, does not produce chemokines for
which CTLs express
receptors [45,46] . Hence optimal effects on
EBV negative HD will likely require the CD30CAR+
CTLs to be further modified with transgenic chemokine receptors
to ensure adequate
accumulation at tumor sites [47] .
In conclusion, the adoptive transfer of EBV-CTLs grafted
with a CAR targeting the CD30
molecule may enhance immunotherapy of patients with EBV+
HD. Acquisition of anti-tumor
effect against CD30+ tumor cells should be benefited by local and
systemic stimulation of the
native receptor by EBV-infected B cells, without harm to the function
of the immune system as a
whole.
http://bloodjournal.hematologylibrary.org/cgi/content/full/blood-2006-11-059139/DC1
We are grateful for the persistent and reliable support of Tatiana
Gotsolva and Fernando
Jimenez of the Flow Cytometry Core Facility (Texas Children’s Hospital,
Houston, TX, USA) for
FACS analyses. We thank Martin Pule, MD (University College London,
London, UK) and Elio
Vanin, PhD (Children’s Memorial Research Center, Chicago, IL, USA)
for assistance in vector
production.
Author contribution:
Barbara Savoldo: designed and performed in vitro and in
vivo experiments, analyzed the data
and wrote the manuscript.
Cliona Rooney: designed experiments and edited the manuscript.
Antonio Di Stasi: performed many of the in vitro experiments.
Hinrich Abken and Andreas Hombach: provided vital reagents for the
research.
Aaron Foster: performed many of the in vivo the experiments.
Lan Zhang: performed many of the in vitro experiments.
Helen Heslop: designed experiments and edited the manuscript.
Malcolm Brenner: designed experiments and edited the manuscript.
Gianpietro Dotti: designed experiments, analyzed the data and wrote
the manuscript.
In this very complicated approach to effective tumor immunotherapy for patients with progressing Hodgkin lymphoma, Barbara Savoldo, Cliona Rooney, Antonio Di Stasi, Hinrich Abken, Andreas Hombach, Aaron Foster, Lan Zhang , Helen Heslop, Malcolm Brenner, and Gianpietro Dotti have added sensitivity from protein antigens to increase the activity of T cells from Hodgkin lymphoma patients. When assayed in immunosuppressed mice, such super-immune T lymphocytes are effective against the B cell-derived neoplastic Reed-Sternberg cells in vivo.
Beyond such protein adjuvants lies the new field of RNA instruction of T cells for tumor immunotherapy as developed by:
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1c. Coughlin CM, Vance BA, Grupp SA, and Vonderheide RH, "RNA-transfected
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1a. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, and Rosenberg SA. "Cancer regression in patients after transfer of genetically engineered lymphocytes". Science. 2006;314:126-129.
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9. Links to Hodgkin Lymphoma Immuno-Pathology:
Links to
Euchromatin Activator RNA Reviews:
Links to
Euchromatin Activator RNA Research:
Links to Ultrastructural
Probes of DNase I-Sensitive Sites:
Links to
RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma
Immuno-Pathology:
Links to Activated
T-Lymphocyte Immunotherapy:
Links to Medical
Systems Biology:
Links to Selective
Gene Transcription:
Links to RNA-Induced
Epigenetics:
Links to RNA-Induced
Embryogenesis:
Links to RNA and
Biological Causality:
Links to Reprogramming
and Neoplasia:
A Brief History of Activator RNA:
"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA". (PowerPoint Presentation).