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GENE THERAPY
From the Center for Cell and Gene Therapy and the
DeBakey Department of Surgery, Baylor College of Medicine, Houston,
Texas.
Primary T cells expressing chimeric receptors specific for tumor or
viral antigens have considerable therapeutic potential. Unfortunately,
their clinical value is limited by their rapid loss of function and
failure to expand in vivo, presumably due to the lack of costimulator
molecules on tumor cells and the inherent limitations of signaling
exclusively through the chimeric receptor. Epstein-Barr virus (EBV)
infection of B lymphocytes is near universal in humans and stimulates
high levels of EBV-specific helper and cytotoxic T cells, which persist
indefinitely. Our clinical studies have shown that EBV-specific T cells
generated in vitro will expand, persist, and function for more than 6 years in vivo. We now report that EBV-specific (but not primary) T
cells transduced with tumor-specific chimeric receptor genes can be
expanded and maintained long-term in the presence of EBV-infected B
cells. They recognize EBV-infected targets through their conventional
T-cell receptor and tumor targets through their chimeric receptors.
They efficiently lyse both. EBV-specific T cells expressing chimeric
antitumor receptors may represent a new source of effector cells that
would persist and function long-term after their transfer to cancer patients.
(Blood. 2002;99:2009-2016) The genetic modification of human T cells to
express tumor antigen-specific chimeric receptors is an attractive
means of providing large numbers of effector cells for adoptive
immunotherapy. The primary mechanisms by which tumor cells escape from
immune recognition, such as down-regulation of major histocompatibility
complex (MHC) molecules, are efficiently bypassed through use of this
strategy. T lymphocytes engineered to express the recombinant receptor
genes are capable of both specific lysis and cytokine secretion on
exposure to tumor cells expressing the requisite target
antigen.1,2
Although adoptive transfer of chimeric receptor-expressing peripheral
blood-derived T lymphocytes has produced some antitumor activity in
mice,3-5 clinical results have been
disappointing.6,7 The most pertinent issue is that
chimeric T cells fail to expand and rapidly lose their function in
vivo. Activation studies performed in transgenic mice suggest that the
function of chimeric receptor proteins depends on the activation status
of the T cell.8,9 Signaling through chimeric T-cell
receptors alone was shown to be insufficient to induce proliferation
and effector function in primary T lymphocytes, unless they had been
prestimulated through their native receptor.8,9 Even under
these conditions, responsiveness was soon lost. This problem is
accentuated by the general lack of tumor cell costimulatory molecules
essential for the induction and maintenance of a T-cell
response.10 The development of strategies to prevent
functional inactivation of chimeric receptor-modified cells in vivo
would greatly enhance their therapeutic value.
Immune regulation of latent EBV infection is one of the best-studied
examples of persistent T cell-mediated immune control. More than 90%
of adults are seropositive for this virus, and their B cells expressing
EBV-encoded latency-associated transforming proteins are tightly
controlled by high levels of EBV-specific HLA-restricted cytotoxic T
lymphocytes (CTLs), which persist indefinitely.11 The
interaction of specific T lymphocytes with the target cells of latent
EBV infection in immunocompetent hosts is characterized by a complex
self-modulating network of cellular immune-mediated interactions,
resulting in potent target cell lysis. These EBV-specific immune
responses can be reconstituted by transfusion of in vitro-generated EBV-specific CTL lines into patients with EBV-associated infections and
malignancies.12-15 The transfused T lymphocytes show a
high initial degree of in vivo expansion and contain all necessary subpopulations to produce regression of even bulky EBV+
tumors. Gene marking studies have demonstrated their persistence for
more than 6 years with retained ability to respond to viral stimulation
in vivo.12-15
The rapid expansion of EBV-specific T cells in vivo and their
persistence in a functional state, lifelong without further immunization, make them attractive candidates for tumor cell targeting via chimeric T-cell receptors (TCRs). We show here that EBV-specific CTLs can be engineered to recognize and lyse tumor cell targets via
chimeric receptors while maintaining their ability to proliferate in
response to EBV target antigens and to destroy virus-infected cells.
Cell lines and antibodies
Construction of chimeric receptor genes
Production of recombinant retrovirus Fresh retroviral supernatants collected from transiently transfected Phoenix-eco cells were used to infect the packaging cell line PG13 in the presence of polybrene (8 µg/mL) twice for 48 hours at 32°C. Viral supernatants were generated on the resulting bulk producer cell lines for 24 hours at 32°C.Generation of EBV-transformed B cell lines Peripheral blood-derived mononuclear cells (5 × 106) were incubated with 10 µL concentrated supernatant from the EBV producer cell line B95-8 in a total of 200 µL medium for 30 minutes. The cells were then plated at 106 cells/well in a flat-bottomed 96-well plate in RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) containing 10% fetal calf serum (FCS; Hyclone, Logan, UT), and 2 mmol/L L-glutamine (Biowhittaker, Walkersville, MD), as well as 1 µg/mL cyclosporin A (Sandoz Pharmaceuticals, Washington, DC). Cells were fed weekly until lymphoblastoid cell lines (LCLs) were established.Generation and transduction of EBV-specific CTL cultures Peripheral blood-derived mononuclear cells (2 × 106) were cocultured with 5 × 104 -irradiated (40 Gy) autologous LCLs per well in a 24-well plate. Starting on day 10, the responder cells were restimulated weekly with
irradiated LCLs at a responder-to-stimulator ratio of 4:1. Two
weekly doses of recombinant human interleukin-2 (IL- 2) (rhIL-2; 40 IU/mL) were added from day 14. Twenty-four hours after the third
stimulation, the cells were transferred to a 24-well plate precoated
with OKT-3 (1 µg/mL; Ortho Pharmaceuticals, Raritan, NJ) and
anti-CD28 antibody (1 µg/mL; Pharmingen, San Diego, CA) at
1 × 106 cells/well and incubated for 48 hours. Cells
were transduced in 24-well plates (Becton Dickinson, Franklin Lakes,
NJ), coated with recombinant FN CH-296 (Retronectin, Takara Shuzo,
Otsu, Japan) at a concentration of 4 µg/cm2. The
prestimulated CTLs were resuspended at 1 × 106 cells/mL
in culture medium containing rhIL-2 (100 IU/mL), and incubated with
equal volumes of freshly generated viral supernatant for 36 hours at
37°C and 5% CO2.
Flow cytometry Cells were stained with fluorescein-conjugated monoclonal antibodies (Becton Dickinson, San Jose, CA) directed against CD3, CD4, CD8, CD16, CD56, and CD25 surface proteins. For each sample, 10 000 cells were analyzed by FACSCalibur with the Cell Quest Software (Becton Dickinson, San Jose, CA). Surface expression of 14.G2a- was analyzed
after incubation of CTLs (1 × 106) with 14.G2a
anti-idiotypic antibody 1A7 (200 ng/5 × 105 cells) in
the presence of normal goat serum for 20 minutes on ice, followed by
incubation with fluorescein isothiocyanate (FITC)-labeled goat
antimouse antibody (Becton Dickinson, San Jose, CA). For tetramer
staining, CTLs from HLA-A2+ donors were incubated with
phycoerythrin (PE)-labeled LMP-2/HLA-A2 tetramer at a final
concentration of 50 mg/mL in phosphate-buffered saline (PBS) plus 2%
FCS for 30 minutes on ice, then washed and stained with peridinin
chlorophyll protein (PerCP)-labeled anti-CD8 antibody for 20 minutes.
One million events were acquired and analyzed.
Measurement of cytokine production Duplicate samples of transduced effector cells (5 × 104/well) were cocultured with various tumor cells or EBV-transformed LCLs at target-to-effector ratios of 3:1 and 1:3 in 96-well round bottomed plates. After 24 hours, the supernatants were harvested and analyzed for human interferon- (IFN-), tumor
necrosis factor- (TNF-), IL-4, IL-10, and IL-12 (Pharmingen) or
granulocyte-macrophage colony-stimulating factor (GM-CSF; R & D Systems, Minneapolis, MN) by ELISA according to the manufacturer's instructions.
Cytotoxicity assays Cytotoxic specificity was determined in standard 51Cr release assays. Various numbers of T-effector cells were coincubated in triplicate with 5000 target cells labeled with 100 µCi (3.7 MBq) 51Cr in a total volume of 200 µL in a V-bottomed 96-well plate. At the end of a 4-hour period at 37°C and 5% CO2, supernatants were harvested, and radioactivity was counted in a gamma counter. Maximum release was determined by lysis of target cells with Triton X. To determine HLA class I or II restriction of cytolysis, target cells were preincubated for 30 minutes with 16.5 ng/mL W6/32 or CR3/43 antibodies (Dako, Carpinteria, CA). For cold target inhibition assays, unlabeled inhibitor cells (cold targets) were seeded in plates at various cold-to-hot target ratios. Effector cells were then added and incubated for 30 minutes at 37°C before labeled target cells (hot targets) were added.Proliferation assays Transduced T lymphocytes were coincubated in triplicate at 5 × 104 cells/well with various tumor cell targets or autologous or allogeneic EBV-LCLs at a 4:1 stimulator-to-responder ratio. Following a 72-hour coincubation period, wells were pulsed with 2.5 µCi (0.0925 MBq) [3H]-thymidine for 18 hours, and the samples were harvested onto glass fiber filter paper for scintillation counting.
Transduced EBV-specific CTLs express the chimeric receptor while maintaining their immunophenotype Eight EBV-specific CTL lines, generated from 4 different seropositive healthy donors13,14 were transduced with 14.G2a- chimeric receptor genes. This receptor is derived from the
14.G2a monoclonal antibody that recognizes GD2, a
ganglioside antigen present on tumors of neural crest
origin,17,22 including neuroblastoma and small cell lung
cancer, as well as glioblastoma and melanoma. Flow cytometric analysis
of CTLs stained with anti-14.G2a idiotype-specific antibody identified
chimeric receptors on 10.2% to 43.1% of the CTLs (mean, 16.5%).
Chimeric receptor expression was maintained over the entire period of
culture (up to 45 days) without any apparent down-regulation.
CD4+ and CD8+ T lymphocytes within the cultured
cell population were transduced equally well (Figure
1).
After 3 stimulations with autologous LCLs, the majority of the CTL
lines had a characteristic immunophenotype12-15:
CD3+ CD8+ on 80% to more than 90% of the
cells and CD3+ CD4+ (T-cell helper) on 4% to
21%. Fewer than 5% of the cells showed an immunophenotype
characteristic of natural killer (NK) cells (CD3 Triggering of the native T-cell receptor but not the transduced and
nontransduced EBV-CTLs in the presence of LCLs, irradiated
G 
Coculture with tumor cells does not result in CTL lysis or growth inhibition To exclude the possibility that coculture with GThe EBV-specific CTLs were not susceptible to cytolysis by JF or LAN-1 tumor cells after coincubation periods of up to 18 hours (not shown). Furthermore, CTL expansion was not decreased in the presence of tumor cells, when compared to allogeneic LCLs or absence of stimulator cells, indicating that the tumor cells did not actively inhibit CTL expansion (not shown). Chimeric receptor-modified CTLs specifically release cytokines in
response to autologous EBV and G The CTL cultures containing 5% to 15% 14.G2a-
Chimeric receptor-modified CTLs specifically lyse autologous
EBV and G -transduced cells. None of the lines had discernible
activity against autologous phytohemagglutinin-stimulated lymphoblasts.
Whereas nontransduced CTLs were incapable of lysing tumor targets
(0%-5%), 21% to 50% (mean, 36.6%) of G-transduced
CTLs. No cytolytic activity of transduced cells against the
G-transduced cells (not shown). The
cytolytic activity of cell lines tested for up to 29 days after
transduction (4 stimulations) was comparable to that of lines tested
over shorter intervals. Retesting of a CTL line after an additional
round of restimulation with autologous LCLs resulted in comparable
cytolytic activity against both EBV and tumor targets (Table 1, CTL
lines 5 and 10).
Figure 4 compares the cytolytic
activities of 3 different 14.2a-
14.G2a- -transduced EBV-specific CTLs was HLA
independent, it could be inhibited by addition of nonlabeled EBV target
cells, whereas lysis of EBV+ targets could be diminished by
competition from nonlabeled tumor cells (Figure
6). Neither allogeneic EBV-LCL nor
G
As an additional demonstration of coexpression of the T-cell receptor,
specific for an EBV-encoded antigen, as well as the 14.G2a-
Chimeric receptor-modified CTLs are rescued to proliferate by stimulation with autologous EBV-LCLs The clinical success of transduced EBV-CTLs will likely depend in part on these cells being able to proliferate when restimulated by autologous EBV targets following exposure to tumor cells. We therefore compared the proliferative responses of the CTLs to tumor targets and to EBV targets after repeated stimulation with either autologous LCLs, allogeneic LCLs, G
Adoptive immunotherapy with chimeric receptor-modified T lymphocytes has shown promise in preclinical studies as a means to combat infectious and malignant diseases.4 However, the first clinical evaluation of chimeric receptor-modified cells revealed a disappointing lack of correlation between in vivo and in vitro cytotoxicity.6,7 One of the major factors limiting successful therapeutic use of modified T cells is their failure to expand and their short life span in vivo, even in the absence of any immune response directed against the chimeric T cells. CD4+ helper function plays a crucial role in establishing or maintaining CD8+ CTL-mediated antiviral or antitumoral immunity,27-29 and long-term maintenance of engineered T cells is clearly improved if both CD8+ and CD4+ transduced T cells are infused, rather than CD8 cells alone.6,7 Previous clinical trials in human immunodeficiency virus (HIV) infection have demonstrated prolonged, high-level persistence of chimeric receptor-modified CD4+ and CD8+ T cells for at least 1 year. However, no significant mean change in plasma HIV RNA or blood proviral DNA was observed in patients with persisting modified T lymphocytes. No analysis of in vivo function of modified T cells over time has been conducted in this study. An explanation for this observation is that even in the continued presence of detectable chimeric receptor-modified cells in vivo, the surviving T lymphocytes may lose their ability to produce cytokines and to lyse their targets, reflecting functional inactivation of the modified cells. Supporting this concept are studies in a transgenic mouse model that showed that chimeric receptor-mediated signaling was not sufficient to trigger activation of resting primary T cells.8,9 Although the lack of coreceptor signaling by most tumor targets probably contributes to this effect,10 it is also likely that chimeric receptors provide only limited access to downstream signaling pathways.8,9 The pattern of T-cell activation triggered by chimeric receptor engagement that we observed in our study, including efficient target cell lysis, reduced levels of specific cytokine release, and lack of cellular proliferation, is reminiscent of the T-cell response to altered peptide ligands as a consequence of incomplete phosphorylation of TCR-associated proximal activation motifs.30,31 Taken together, the above results indicate that efficient and sustained
expansion and activation of transfused chimeric effector T lymphocytes
in vivo will require (1) T-cell helper activity provided in a cognate
fashion, (2) signaling through the native TCR/CD3 complex, and (3) the
presence of costimulatory signals and cytokines. Our results suggest
that the introduction of chimeric receptors into ex vivo-generated
EBV-specific T-cell lines will meet all of these requirements (Figure
9). These cell lines contain antigen-specific CD4+ helper T cells that contribute to
immune control of EBV latency by providing growth factors capable of
maintaining both CD4+ and CD8+ cells, as well
as CD8+ cytotoxic T cells.14 The target cells
are EBV+ B lymphocytes, which present antigens extremely
well. They express both class I and class II MHC-restricted antigenic
epitopes, facilitating cognate interactions between CD44
and CD8+ T cells, and are rich in costimulator molecule
expression.12-15
What is the evidence that the properties of such T-cell lines will be reiterated in vivo? In patients given gene-marked EBV-specific CTLs, we can detect a high degree of in vivo expansion, resulting in long-term persistence and antiviral activity for more than 6 years.12-15 Expression of chimeric receptor genes in EBV-specific CTLs does not interfere with the ability of the cells to proliferate or to respond to autologous EBV-infected targets (Figure 2A,B). Their ability to kill tumor cell targets through the chimeric receptor is retained even after expansion driven through the EBV-antigen specific native receptors (Table 1, Figure 4). Following exposure to tumor cells in culture, transduced CTLs can be rescued to proliferate and expand by stimulation through their EBV-specific receptor (Figure 8). In our system, none of the tumor cell targets proved toxic to the CTLs or inhibitory to their expansion. We therefore postulate that as a result of the continued presence of viral antigens in an EBV-infected host, engineered antitumor T lymphocytes with native specificity for EBV antigens will survive for extended periods. Such an effect can result only if the same T cell is both EBV and tumor specific, a requirement that was met in the present study, both phenotypically (as demonstrated by fluorescent analysis with an anti-idiotype monoclonal antibody and an EBV tetramer) and functionally (as shown by cross-inhibition of each target cell with either EBV-infected B lymphocytes or tumor cells). Although cross-inhibition can be demonstrated in short-term assays in vitro, it should not prove a significant limitation in vivo because a single T cell can kill multiple cellular targets sequentially, disengaging from each once killing has been achieved. This effect is illustrated by the ability of chimeric EBV-CTLs that have been repeatedly stimulated by EBV-LCLs, to subsequently kill tumor targets (Figure 3). Hence, after infusion of chimeric EBV-specific T cells into EBV+ individuals, there should be lifelong in vivo restimulation via the native T-cell receptor in the presence of adequate costimulation as illustrated in Figure 6. This will prevent functional inactivation of the cells and should enable them to continuously lyse any chimeric receptor target cells they encounter. Without performing a clinical study, we cannot be certain that there will be sufficient stimulation by EBV antigens in vivo to produce expansion and maintenance of chimeric T cells at a sufficient level to produce antitumor effects. Nonetheless, recent evidence from our current adoptive transfer studies of EBV CTLs suggests that expansion and functional maintenance of these cells will occur even in patients with "normal" levels of EBV DNA and without evident EBV+ malignancy (C.R. and Barbara Savoldo, unpublished observations, August 2001). This effect is probably a consequence of periodic reactivation of EBV (and other herpesviruses). Serologic evidence suggests that reactivation after primary infection is a frequent event,32 and recent measures of serum EBV-DNA levels have produced similar results.33,34 Should the number of functionally activated EBV-specific CTLs and their level of activation in the absence of massive EBV reactivation prove to be too low to provide a stimulus of sufficient strength for chimeric-mediated tumor cell lysis, it may prove possible to boost T-cell responses by immunization with autologous irradiated LCLs. In principle, the above strategy may be used for any tumor target to
which a chimeric receptor can be made. However, one of the major
advantages of chimeric T cell-based therapies is that they obviate the
need to select and expand the scanty tumor-specific T cells present in
the circulation. The EBV-specific CTL chimeras we describe here would
seem to remove that advantage, because an antigen-specific selection
and expansion process will be required after all. But, in practical
terms, the expansion of EBV CTLs and the expansion of tumor-specific
CTLs are 2 quite separate propositions. The high frequency of
EBV-specific precursor cells in peripheral blood and the excellent
antigen-presenting capacity of EBV-infected B cells makes this a robust
system. Over the past 6 years, we have successfully generated
EBV-specific CTL lines from 138 of 140 donors, including cancer
patients pretreated with chemotherapy.35 More than 100 patients with EBV-associated infections or malignancy have received
EBV-CTL infusions with no serious adverse effects. These results have
been confirmed by others.36 Moreover, because the infused
cells are expected to expand markedly in vivo and persist in the
circulation for extended periods, only limited numbers of cells may
need to be grown and infused.12-15 The use of EBV-specific
cell lines is to be preferred to the use of clones because (1) they are
simpler to prepare, (2) the combination of CD4 and CD8 cells present
produces a more sustained immune response than CD8+ clones
alone,12,14,24 and (3) EBV antigen escape mutants are less
likely to arise.23 Because high-efficiency cytolysis was
achieved with cultures containing up to 90% nontransduced CTLs, there
would be no need to coexpress marker or selection genes
Submitted May 3, 2001; accepted November 16, 2001.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Malcolm K. Brenner, Center for Cell and Gene Therapy, 6621 Fannin St, MC3-3320, Houston, TX 77030; e-mail: mbrenner{at}bcm.tmc.edu.
1.
Eshhar Z, Waks T, Gross G, Schindler DG.
Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors.
Proc Natl Acad Sci U S A.
1993;90:720-724 2. Stancovski I, Schindler DG, Waks T, Yarden Y, Sela M, Eshhar Z. Targeting of T lymphocytes to Neu/HER2-expressing cells using chimeric single chain Fv receptors. J Immunol. 1993;151:6577-6582[Abstract]. 3. Altenschmidt U, Klundt E, Groner B. Adoptive transfer of in vitro-targeted, activated T lymphocytes results in total tumor regression. J Immunol. 1997;159:5509-5515[Abstract].
4.
Hwu P, Yang JC, Cowherd R, et al.
In vivo antitumor activity of T cells redirected with chimeric antibody/T-cell receptor genes.
Cancer Res.
1995;55:3369-3373 5. McGuinness RP, Ge Y, Patel SD, et al. Anti-tumor activity of human T cells expressing the CC49-zeta chimeric immune receptor [see comments]. Hum Gene Ther. 1999;10:165-173[CrossRef][Medline] [Order article via Infotrieve].
6.
Mitsuyasu RT, Anton PA, Deeks SG, et al.
Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects.
Blood.
2000;96:785-793
7.
Walker RE, Bechtel CM, Natarajan V, et al.
Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection.
Blood.
2000;96:467-474
8.
Brocker T, Karjalainen K.
Signals through T cell receptor-zeta chain alone are insufficient to prime resting T lymphocytes.
J Exp Med.
1995;181:1653-1659
9.
Brocker T.
Chimeric Fv-zeta or Fv-epsilon receptors are not sufficient to induce activation or cytokine production in peripheral T cells.
Blood.
2000;96:1999-2001
10.
Krause A, Guo HF, Latouche JB, Tan C, Cheung NK, Sadelain M.
Antigen-dependent CD28 signaling selectively enhances survival and proliferation in genetically modified activated human primary T lymphocytes.
J Exp Med.
1998;188:619-626 11. Khanna R, Burrows SR. Role of cytotoxic T lymphocytes in Epstein-Barr virus-associated diseases [in process citation]. Annu Rev Microbiol. 2000;54:19-48[CrossRef][Medline] [Order article via Infotrieve]. 12. Heslop HE, Ng CY, Li C, et al. Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nat Med. 1996;2:551-555[CrossRef][Medline] [Order article via Infotrieve]. 13. Rooney CM, Smith CA, Ng CY, et al. Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation. Lancet. 1995;345:9-13[CrossRef][Medline] [Order article via Infotrieve]< |
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Abstract
Introduction
)
CD16+ CD56+). One cell line
was predominantly CD3+ CD4+. Transduction of
CTLs did not result in any changes of cellular immunophenotypes by
comparison with nontransduced cells (not shown). Hence, introduction
and expression of the chimeric receptor does not shift the phenotype of
EBV-specific CTLs from that known to be able to expand, persist, and
induce tumor regression following infusion in vivo.
did not release cytokines in response to incubation
with G
a major source
of immunogenicity after chimeric T-lymphocyte infusion. The individual
components of this proposed system have already been safely used in
humans; the chimeric receptor in the form of monoclonal antibodies
administered to patients with malignancy,