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Prepublished online as a Blood First Edition Paper on July 5, 2002; DOI 10.1182/blood-2002-04-1041.
GENE THERAPY
From the Cancer Immunology Program, Sir Donald and Lady
Trescowthick Laboratories, Peter MacCallum Cancer Institute, and the
Rotary Bone Marrow Research Laboratory, Royal Melbourne Hospital
Research Foundation, Victoria, Australia.
Tumor cells are usually weakly immunogenic as they largely express
self-antigens and can down-regulate major histocompatability complex/peptide molecules and critical costimulatory ligands. The
challenge for immunotherapies has been to provide vigorous immune
effector cells that circumvent these tumor escape mechanisms and
eradicate established tumors. One promising approach is to engineer T
cells with single-chain antibody receptors, and since T cells require 2 distinct signals for optimal activation, we have compared the
therapeutic efficacy of erbB2-reactive chimeric receptors that contain
either T-cell receptor zeta (TCR- The natural recognition and elimination of cancers
by the adaptive arm of the immune system often fails and approaches to stimulate this adaptive antitumor immunity can be severely limited by
tumor escape mechanisms.1-3 One promising approach
conceived almost a decade ago was the genetic modification of T cells,
resulting in engraftment with integral membrane single-chain Fv (scFv)
chimeric signaling receptors, reactive with tumor-associated
antigens.4,5 Such T cells have been shown to mediate tumor
antigen-specific inhibition of tumor growth in vitro and in vivo, in a
major histocompatability complex (MHC) class I-independent
manner.6,7 Our recent study compared scFv-Fc CD28 provides possibly the most potent comitogenic signal, functioning
in synergy with the TCR, as a general amplifier of early TCR signal
transduction and modulating the signaling environment around the site
of TCR engagement.10,11 Costimulation of T cells via CD28
following ligation with tumor cell-surface CD80 and CD86 molecules or
bispecific antibody (Ab)-mediated cross-linking to
tumor-associated antigen (TAA) has been demonstrated to greatly stimulate subsequent tumor rejection and T-cell memory in
vivo.12,13 Chimeric receptors incorporating CD28 have been
shown to mediate enhanced T-cell activation in vitro.14-19
Given that many tumor cells do not express costimulatory
ligands,20 it was not clear whether this approach was
sufficient to endow primary T lymphocytes with antitumor efficacy in
vivo. Despite their promise, T lymphocytes engineered with scFv
chimeras have had limited efficacy against established disease in
mouse tumor models.7 Importantly, we now demonstrate
that providing antigen-specific costimulation to gene-modified primary
mouse T cells using CD28-containing scFv chimeric receptors can
effectively eliminate established tumors using interferon gamma
(IFN- Cell culture
Mice
Chimeric receptor gene construction A 767-bp fragment of DNA coding for scFv of anti-erbB2 (a kind gift from Dr Winfried Wels, Institute of Experimental Cancer Research, Germany)21 and a marker epitope from c-myc was amplified by polymerase chain reaction from the pSW50-5 vector and subcloned into XbaI/BstEII-digested pRSVscFv R
(a kind gift from Zelig Eshhar, Weizmann Institute, Rehovot, Israel).
The chimeric gene constructs were composed of the scFv-anti-erbB2
monoclonal antibody (mAb), a membrane proximal hinge region of human
CD8, and the transmembrane and cytoplasmic regions of the human TCR-
chain (scFv-anti-erbB2-) or the transmembrane and cytoplasmic
regions of the mouse CD28 signaling chain fused to the cytoplasmic
region of TCR- (scFv-anti-erbB2-CD28-) (Figure
1A). For detection purposes each receptor
contained a c-myc tag epitope at the C-terminus of the
VL region. The scFv-anti-erbB2 chimeric receptors were
digested with SnaB1/XhoI and subcloned into the
HpaI/XhoI restriction sites of the retroviral
vector, pLXSN (a kind gift from Dusty Miller, Fred Hutchinson Cancer
Research Centre, Seattle, WA) containing the long terminal repeat and a neomycin resistance gene under the control of an SV40
promoter.
Retroviral gene transfer of mouse spleen T lymphocytes Stable GP+E86 ecotropic packaging cell lines expressing the scFv-anti-erbB2- or scFv-anti-erbB2-CD28- receptors were
isolated as described previously.6,7 GP+E86 clones
producing approximately 107 cfu/mL were used for
transduction of mouse spleen T lymphocytes. Spleen cells from mice were
initially depleted of red blood cells (RBCs) by hypotonic lysis with
NH4Cl and enriched by passing through a nylon wool syringe
as described previously.7 Enriched T lymphocytes (107) were then cocultivated for 72 hours with
5 × 105 viral-producing packaging cells in DMEM
supplemented with 4 µg/mL polybrene, 5 µg/mL phytohemaglutinin
(PHA) (Sigma, St Louis, MO), and 100 U/mL rIL-2. Following
cocultivation, T cells were separated from adherent packaging cells,
washed with DMEM, and cultured in DMEM supplemented with 100 U/mL
rIL-2. T cells were subsequently analyzed for transduction efficiency
by flow cytometry and used for in vitro and in vivo experiments.
Consistent with previous observations,6 the majority of T
cells selected by the transduction procedure were CD8+ (> 85% TCR +CD8+,  6%
TCR+CD4+) (data not shown). In some
experiments, transduced T cells were treated with anti-mouse CD4
Ab-conjugated immunomagnetic beads (GK1.5, Miltenvi Biotec, Bergisch
Gladbach, Germany). CD4+ T cells were depleted using a MAC
separator according to the supplier's specifications. Depletion (6%
to 0% CD4+) was verified by flow cytometry.
Flow cytometry Detection of cell-surface chimeric receptor expression on mouse T lymphocytes was achieved by indirect immunofluorescence with a c-myc tag Ab purified from supernatants of mouse 9E10 cells,22 followed by staining with a phycoerythrin (PE)-labeled anti-mouse Ig mAb (Beckon Dickinson, San Jose, CA). Background fluorescence was assessed using a purified IgG1 isotope Ab (3S193; Ludwig Institute for Cancer Research, Melbourne, Australia). Cell-surface phenotyping of transduced cells was determined by direct staining with Quantum-Red-labeled anti-TCR (clone H57-597;
PharMingen, San Diego, CA); fluorescein isothiocyanate (FITC) anti-CD4
(RM4-5; PharMingen); and Quantum-Red-labeled anti-CD8 (R-3762; Sigma)
mAbs as previously described.6 Cell-surface phenotyping of
tumor cell lines was determined by indirect immunofluorescence with
anti-human erbB2 (9G6.10, Neomarkers, Fremont, CA), anti-mouse or
anti-human CD80 (mouse, 1G10; human, BB1; Sigma), and CD86 (mouse,
GL1; human, 2331[FUN-1]; Sigma) mAbs, followed by staining with a
fluorophore-labeled anti-Ig mAb.
Antigen-specific binding, cytotoxicity, and cytokine secretion The binding capacity of gene-modified mouse T lymphocytes was determined in a rosetting assay as described.7 The cytolytic capacity of transduced T cells was determined in a 6-hour 51Cr-release assay. Mouse IFN-, IL-2, granulocyte
macrophage-colony-stimulating factor (GM-CSF), tumor necrosis
factor alpha (TNF), IL-4, and IL-10 secretion by scFv-modified mouse
T lymphocytes after erbB2 antigen ligation was detected by
enzyme-linked immunosorbent assay (ELISA). Transduced T cells
(106) (transduced with LXSN plus
scFv-anti-erbB2-, scFv-anti-erbB2-CD28-, or
mock-transduced T cells) were cultured with 106
erbB2+ (Lovo, COLO 205, MDA-MB-435 or MC-38-erbB2) or
erbB2 (24JK or MC-38) tumor cells in 12-well plates for
20 hours. No exogenous IL-2 was added. Cytokine production through
stimulation of endogenous CD3 and CD28 receptors was assessed for each
T-effector cell population using soluble CD3 (1 µg/mL) (145.2C11;
PharMingen) and CD28 (1 µg/mL) (37.51; PharMingen) mAbs. Following
incubation, supernatants were harvested and the level of cytokine
production was measured by ELISA (PharMingen) according to the
supplier's specifications.
Proliferation assays Proliferation assays were performed in 96-well U-bottom plates. The scFv-modified mouse T lymphocytes (105 cells/well, 5 × 104 cells/well, or 104 cells/well) were cultured in media alone, cocultured with irradiated MC-38-erbB2 or MC-38 tumor cells (105 cells/well), or stimulated with plate-bound CD3 plus CD28 mAb (1 µg/mL) for 3 days. Cultures were pulsed with 0.5 µCi/well (0.0185 MBq) of [3H]-thymidine (Amersham, Aylesbury, United Kingdom) for the last 16 hours of assay. No exogenous IL-2 was added. Incorporation of radioactivity was measured in a TRI-CARB 2100TR Liquid Scintillation Counter (Packard, Meriden, CT).Adoptive transfer models The antitumor response of transferred T cells was assessed against 2 different erbB2+ tumor cell lines following subcutaneous inoculation. First, 106 mouse 24JK sarcoma cells and/or 5 × 106 human COLO 205 colon carcinoma cells were injected subcutaneously into opposite flanks of groups of 5 to 10 scid mice. Spleen T lymphocytes from BALB/c mice (transduced with LXSN plus scFv-anti-erbB2-, scFv-anti-erbB2-CD28-, or
mock-transduced T cells) were injected intravenously into groups of 10 scid mice at 6 hours (day 0, 5 × 106) and 24 hours (day
1, 5 × 106), on day 3 (107), or used
for titration experiments at day 1 (105, 106,
or 107) after tumor inoculation. In addition, adoptive
transfer of scFv-transduced spleen T lymphocytes
(5 × 106, day 0 and day 1) from BALB/c
pfp/, BALB/c IFN-/, or BALB/c
pfp/IFN-/ mice were used to evaluate
involvement of pfp and IFN-. In the second model,
5 × 106 MC-38 and/or MC-38-erbB2 tumor cells were
injected subcutaneously into opposite flanks of groups of 5 to 10 scid
mice. Spleen T lymphocytes from BALB/c mice (transduced with LXSN plus
scFv anti-erbB2-, scFv-anti-erbB2-CD28-, or mock-transduced T
cells) were injected intravenously into groups of 10 scid mice at 6 hours (day 0, 5 × 106) and 24 hours (day 1, 5 × 106) or on day 3 (107) after tumor
inoculation. Subsequent tumor growth was monitored daily and measured
by a caliper square along the perpendicular axes of the tumors. The
data were recorded as the mean tumor size (mm2, product of
the 2 perpendicular diameters) ± SEM.
Experimental pulmonary metastasis model Scid mice were injected intravenously with 5 × 106 human MDA-MB-435 breast carcinoma cells to establish pulmonary metastases. Spleen T lymphocytes (107) from BALB/c mice (transduced with LXSN plus scFv-anti-erbB2-, -CD28-, or mock-transduced T cells) were injected intravenously into
groups of 10 mice at day 1, 5, or 10 after tumor inoculation. In
addition, adoptive transfer of scFv-transduced spleen T lymphocytes (107, day 1) from BALB/c pfp/, BALB/c
IFN-/, or BALB/c
pfp/IFN-/ mice were used to evaluate
involvement of pfp and IFN-. Mice were monitored daily for tumor
growth, indicated by respiratory distress and loss of body condition.
There were 3 parameters of tumor growth, determined as follows: (1) in
survival experiments, mice that were morbid were killed and the day of
death recorded; (2) some groups of mice were killed at day 10 or 18 (when those not receiving T cells were morbid) and whole lung weight
(g) was recorded; or (3) some groups of mice were killed at day 10 or 18 and harvested lungs were fixed in 10% formalin, embedded, sectioned and stained with hematoxylin and eosin (H&E) for histologic examination.
Expression of the chimeric scFv-anti-erbB2- chimeras in Jurkat T cells (with
specificities for different antigens) produced maximal levels of IL-2
in response to specific antigens compared with stimulation via either
receptor alone.24 To avoid having to coexpress 2 receptors
in T cells, we created a series of scFv (VH and
VL) anti-erbB2 chimeric receptors containing the
intracellular domains of CD28 with Fc RI- or TCR- in series.
Initially, chimeras were chosen following successful transient
transfection in COS-7 cells and these were then stably expressed in
Jurkat T cells. Jurkat T cells transduced with chimeras containing the
transmembrane and cytoplasmic domains of CD28 fused to the TCR-
signaling moiety (Figure 1A) produced more IL-2 than Jurkat T cells
equivalently expressing the scFv- chimera (data not shown). These
data were very encouraging; however, the effectiveness of this type of
chimeric receptor needed to be tested in primary T lymphocytes, in
vivo. Both receptor gene constructs were subcloned into the retroviral
vector pLXSN and high titer virus-producing GP+E86 clones were used to
transduce enriched naive T lymphocytes from BALB/c mouse spleens as
previously described.6 High and equivalent levels of
expression of the scFv-anti-erbB2- and -CD28/ chimeric receptors
were reproducibly detected on T cells (Figure 1B; n = 5).
Antigen-specific, MHC-independent lysis, enhanced cytokine production, and T-cell proliferation We next sought to compare the activity of each cytoplasmic domain in stimulating T-cell function against erbB2+CD80CD86 tumor target
cells. The cytolytic capacities of T cells expressing either the
scFv-anti-erbB2- receptors (T-scFv- cells) or
scFv-anti-erbB2-CD28- receptors (T-scFv-CD28- cells) were
evaluated in standard 6-hour 51Cr release assays. T cells
expressing either receptor were engrafted with the equivalent ability
to specifically conjugate to (binding assays, data not shown) and lyse
the erbB2+ human COLO 205 colon carcinoma, MDA-MB-435
mammary carcinoma, and mouse MC-38-erbB2 colon adenocarcinoma cell
lines (Figure 2A). Equivalent levels of
cytolysis were also mediated by both transduced T-effector cell
populations after 16 hours (data not shown). Lysis of the erbB2
antigen-negative 24JK mouse sarcoma or MC-38 cell lines was not
detected, demonstrating the antigen-specificity of cytolysis (Figure
2A). Overall, the data indicated that the scFv-CD28- chimera was
functional, but that cytolytic function was neither enhanced nor
diminished by fusing CD28 and cytoplasmic domains.
One of the major consequences of CD28-mediated signaling is the
increased production of T-cell cytokines.25 We therefore compared the capacity of T-scFv- Inhibition of colon carcinoma by scFv-CD28- and scFv-anti-erbB2-CD28- chimeric
receptors to stimulate optimal T-cell antitumor function was evaluated
in adoptive transfer assays using tumor-bearing scid mice. Transduced
T-scFv- or T-scFv-CD28- cells were adoptively transferred
intravenously into scid mice 6 hours and 1 day after the subcutaneous
inoculation of these mice with erbB2+ COLO 205 or MC-38
tumor in the right flank and erbB2- 24JK or MC-38 tumor,
respectively, in the left flank. Both transduced T-cell populations
were capable of mediating a significant antigen-specific antitumor
response against the erbB2+ tumors, but not the
erbB2 tumors (Figure 3A).
Importantly, T-scFv-CD28- cells demonstrated complete eradication of
7 of 10 COLO 205 tumors (Figure 3A) and 8 of 10 MC-38-erbB2 tumors
(Figure 3A). Given the effectiveness of early treatment of the human
and mouse colon adenocarcinomas with T-scFv-CD28- cells, we next
compared the antitumor efficacy of one dose of T cells (107
cells) against 3-day established COLO 205 tumors (mean size ~ 8 mm2). Although no complete tumor eradications were
observed, statistically greater inhibition of growth of
erbB2+ COLO 205 tumors was observed in mice injected with
T-scFv-CD28- cells compared with T-scFv- cells (Figure 3B).
Similar data were obtained with T-cell transfers into scid mice with
established MC-38-erbB2 tumors (data not shown). To further compare the
potency of T-scFv-CD28--mediated response(s) in vivo we assessed
the antitumor efficacy of different doses of T-scFv- cells or
T-scFv-CD28- cells (107, 106, or
105) injected into groups of 10 scid mice at day 1 after
COLO 205 tumor inoculation (Figure 3C). Impressively, T-scFv-CD28-
cells were at least 10-fold more potent than T-scFv- cells in
inhibiting tumor growth (P < .01), and 106
T-scFv-CD28- cells eradicated tumors in 5 of 10 mice compared with 2 of 10 mice injected with 107 T-scFv- cells (Figure 3C).
The greater antitumor capacity of T-scFv-CD28- cells was clearly
evident from 3 independent experiments where collectively a total of 30 mice were inoculated with either 107 T-scFv-CD28- cells
or 107 T-scFv- cells (T-scFv-CD28- cells: 25 tumor-free versus T-scFv- cells: 8 tumor-free,
P < .0001).
Perforin and IFN- cells were so effective in vivo,
we next sought to evaluate the relative importance of their cytotoxicity and IFN- secretion in controlling tumor growth. T cells
from BALB/c wild-type (WT), IFN-/, perforin
(pfp)/, and
pfp/IFN-/ mice were transduced with
the scFv-CD28- or scFv- chimeric receptors. Importantly,
expression of the scFv-CD28- (Figure 4A) and scFv- (data not shown)
chimeras in T cells from gene-targeted mice was equivalent to that
detected in WT T cells. Phenotypic characterization of the
scFv-CD28--transduced T cells from all mouse strains confirmed that
only pfp/ and
pfp/IFN-/ T cells had defective
cytolytic activity against the MC-38-erbB2+ tumor cells
(Figure 4B). Transduced pfp/ T cells retained a similar
capacity to produce IFN- as WT T cells, following antibody-mediated
stimulation of endogenous CD3 and CD28 receptors (data not shown). The
proliferative ability of T cells from all 3 mutant mouse strains,
following chimeric receptor ligation by erbB2 antigen or stimulation of
endogenous CD3 and CD28 receptors, was observed to be at least
equivalent to that of transduced WT T cells (Figure 4C). Having
established the in vitro functional capacity of transduced T cells from
each of the mutant strains of mice, the ability of these T cells to mediate in vivo tumor protection was compared against the antitumor activity of WT T cells.
Transduced T cells from WT, IFN-
IFN- chimera to confer to
T cells antitumor activity against established subcutaneous tumors,
including MDA-MB-435 (data not shown), we next tested efficacy of
transduced T cells against established human MDA-MB-435 breast
carcinoma metastases in scid mice. Chimera-transduced T cells were
adoptively transferred intravenously into scid mice on day 1, 5, or 10 after the intravenous inoculation of MDA-MB-435 tumor cells. The
MDA-MB-435 tumor cells effectively lodge in the lung and grow rapidly,
killing the mice within 16 to 20 days of inoculation. Adoptive transfer
of T-scFv-CD28- cells one day after tumor inoculation rescued 80%
of mice (8 of 10), compared with 20% survival of mice receiving
T-scFv- cells (Figure 6A). The delay
of T-cell transfer until day 5 reduced the number of tumor eradications
(60% survival) mediated by T-scFv-CD28- cells (Figure 6B).
T-scFv- cells were partially effective, prolonging survival of 20%
of mice beyond that observed for animals receiving mock-transduced T
cells or no T-cell treatment; however, all the mice in this group did
succumb to tumor (Figure 6B). Just as observed in the xenogeneic COLO
205 tumor model, the enhanced IFN- production by T-scFv-CD28-
cells was absolutely critical for the superior antitumor activity of
these effector cells against one-day established tumors, since the
MDA-MB-435 tumors grew unaffected in mice that received
IFN-/T-scFv-CD28- cells (Figure 6C).
Interestingly, in this model pfp played a reduced role in the
T-scFv-CD28- cell-mediated antitumor response(s) since the transfer
of pfp/T-scFv-CD28- cells still rescued 70% of
mice.
It must be noted that few immunotherapies have ever been described that
can eliminate established solid tumors or metastases, even in mouse
experimental models. Most dramatic was the potency of 107
T-scFv-CD28-
The transfer of specific chimeric antigen-recognition receptors
into T cells offers great potential to target T cells to any tumor-associated antigen of interest. Specifically, redirecting T cells
using scFv of antibody-chimeric receptors enables the efficient binding
of effector T cells to tumors in a non-MHC-restricted fashion, thus
bypassing the MHC/peptide complex loss that is a major escape mechanism
for most tumors.27 Nevertheless, previous scFv-chimeric
receptor designs that incorporated TCR- A most striking observation from our studies using T cells from
gene-targeted mice was that the superior antitumor efficacy of the
scFv-CD28- Another important observation in our study was the equivalent cytokine
secretion triggered by the scFv-CD28- It is notable that even the most successful recent studies that have
redirected T-cell specificity using scFv chimeras7,30 or
TCR genes42,47 have protected against minimal tumor
burden. By contrast, we have successfully retrovirally infected the
majority of primary mouse T cells and demonstrated significant efficacy of these T-scFv-CD28- Despite encouraging clinical results for cellular immunotherapies using
LAK cells and TILs,48 their general application to all
cancers has been hampered by lack of specificity, poor homing
capabilities, and difficulty in isolating a sufficient number of these
cells. Redirected CTL therapy involving expression of chimeric
receptors has several advantages over other immunotherapies due to (1)
abundance of naive T cells available for gene transfer, (2) variety of
TAA expressed on a broad spectrum of tumors, and (3) MHC-unrestricted
reactivity of scFv-chimeric receptors. Although in many instances the
MHC/peptide may be a more specific target than a TAA, many tumors
readily down-regulate the expression of MHC/peptide complexes. While
effective gene-delivery and autoimmunity remain hurdles with redirected
CTL approaches, rapid advances in gene transfer technology and
humanization of vectors coupled with the encouraging in vivo data
presented herein compel further translation of the scFv approach to
eventual clinical trials of safety and efficacy. Our ultimate goal of
optimizing the signaling capacity of scFv-chimeric receptors has been
to significantly enhance the specificity and potency of antitumor T
cells. Our demonstration of the efficacy and effector mechanisms
provided by a novel scFv-CD28-
The authors wish to thank Dr Ian Davis for his critical reading of this manuscript and the staff of the Peter MacCallum Cancer Institute animal facilities for the caring and maintenance of mice used in this study.
Submitted April 4, 2002; accepted June 17, 2002.
Prepublished online as Blood First Edition Paper, July 5, 2002; DOI 10.1182/blood-2002-04-1041.
Supported by a program grant from the Susan G. Komen Breast Cancer Foundation and the Anti-Cancer Council of Victoria. M.J.S. is currently supported by a National Health and Medical Research Council of Australia (NH&MRC) Principal Research Fellowship.
M.J.S. and P.K.D. contributed equally to this work as senior authors.
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: Phillip Darcy, Cancer Immunology, Peter MacCallum Cancer Institute, Locked Bag 1, A'Beckett St, Victoria, Australia, 8006; e-mail: p.darcy{at}pmci.unimelb.edu.au.
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Top
Abstract
Introduction
) or scFv-CD28-
) chimeric receptors equivalently lysed the erbB2+
tumors COLO 205, MDA-MB-435, or MC-38-erbB2, but not the
erbB2
) were unable to lyse the
erbB2
) or -CD28-
) or pLXSN encoding the scFv-anti-erbB2-
) T-scFv-anti-erbB2-
)
T-scFv-anti-erbB2-CD28-
). For all experiments, results are represented as
the mean tumor size (mm2) ± SEM. Arrows depict the
days of T-cell transfer. COLO 205 tumor growth inhibited by
T-scFv-CD28-
