|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1706-1715
Natural Killer Cell-Mediated Eradication of Neuroblastoma Metastases
to Bone Marrow by Targeted Interleukin-2 Therapy
By
Holger N. Lode,
Rong Xiang,
Torsten Dreier,
Nissi M. Varki,
Stephen D. Gillies, and
Ralph A. Reisfeld
From The Scripps Research Institute, Department of Immunology; the
University of California, Cancer Center, La Jolla, CA; and Fuji
ImmunoPharmaceuticals Corp, Lexington, MA.
 |
ABSTRACT |
Targeted interleukin-2 (IL-2) therapy with a genetically engineered
antidisialoganglioside GD2 antibody-IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of
neuroblastoma. The mechanism involved is exclusively natural killer
(NK) cell-dependent, because NK-cell deficiency abrogated the
antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell-deficient mice or
immunocompetent mice depleted of CD8+ T cells in vivo. A
strong stimulation of NK-cell activity was also shown in vitro.
Immunohistology of the leukocytic infiltrate of livers from treated
mice revealed a strong staining for NK cells but not for
CD8+ T cells. The therapeutic effect of the fusion
protein was increased when combined with NK-cell-stimulating agents,
such as poly I:C or recombinant mouse interferon- . In
conclusion, these data show that targeted delivery of cytokines to the
tumor microenvironment offers a new strategy to elicit an effective
cellular immune response mediated by NK cells against metastatic
neuroblastoma. This therapeutic effect may have general clinical
implications for the treatment of patients with minimal residual
disease who suffer from T-cell suppression after high-dose chemotherapy
but are not deficient in NK cells.
| |
INTRODUCTION |
MORE THAN 50% of the patients with stage
4 neuroblastoma initially present with disseminated metastasis to
distant organ sites, predominantly bone marrow, making effective
treatment for this disease most challenging. Drastic therapeutic
interventions with radiotherapy and/or high-dose chemotherapy,
followed by allogeneic or autologous bone marrow
transplantation,1 have resulted in only minor improvements
in the overall survival rate of such patients. Recent encouraging
results from phase I and I/II clinical trials using adjuvant therapy
with murine (14G2a) and human/mouse chimeric (ch14.18) monoclonal
antibodies (MoAbs) directed against the disialoganglioside GD2 showed more than 50% response rates in these patients,
including several long-term and complete remissions.2-5
Other than their use in the treatment of neuroblastoma and a few other
malignancies, eg, colon carcinoma,6 the therapeutic
application of unmodified MoAbs has enjoyed only limited success in the
clinical setting, despite their extensively documented unique targeting
abilities in vivo and in vitro. Effector functions elicited by MoAbs
include complement and antibody-dependent cellular cytotoxicity, the
latter mediated primarily by such Fc receptor-bearing effector cells as monocytes, macrophages, and natural killer (NK)
cells.7,8 A strategy to increase the therapeutic efficiency
of MoAbs is to additionally stimulate effector cells by means of
immunomodulators, eg, recombinant interleukin-2 (rIL-2). This cytokine
stimulates a broad range of immune cells, including T and B cells,
monocytes, macrophages, and NK cells. Furthermore, rIL-2 was also shown
to generate unique activated lymphocyte populations of NK-cell
phenotype, ie, lymphokine activated killer (LAK) cells9,10
in vitro and in vivo, which effectively mediate antibody-dependent
cellular cytotoxicity.11 Therefore, systemic IL-2, combined
with LAK cells, was applied in clinical trials that produced
encouraging antitumor responses in some patients with
melanoma12 and renal cell carcinoma.13 In
contrast, in neuroblastoma such systemic and nonspecific IL-2
treatments resulted in only modest regressions of
metastases.14 The promising data from clinical trials with anti-GD2 MoAbs, per se, and the experience from systemic
IL-2 therapies led to a first phase I/Ib trial.15 In this
study of the Children's Cancer Group, 31 patients with refractory
neuroblastoma received IL-2 and 14.G2a and were monitored for
toxicities and responses to therapy. One patient had a partial response
with a reduction of a large retroperitoneal mass decreasing in size by
75%. A reduction in bone marrow metastases was detectable in three
additional patients.
The tumor-specific delivery of immunomodulators, in contrast to
systemic delivery, achieves high cytokine concentrations within the
tumor microenvironment that effectively stimulate cellular immune
responses against syngeneic malignancies. This approach can be
accomplished by either cytokine gene therapy or antibody-cytokine fusion proteins. The first approach uses patient-specific, ex vivo
genetic modification of autologous tumor cells or fibroblasts to
express various cytokines,16 inducing a local inflammatory response capable of mediating a systemic immune response effective against distant metastases. However, patients who are subjected to such
immunotherapeutic approaches are frequently pretreated by standard
radio-/chemotherapy protocols followed by bone marrow transplantation,
which are known to be highly immune suppressive and adversely affect
the T-cell-dependent immune system. This therapy is even more severe
in cases involving allograft transplantation, because the prevention of
graft-versus-host disease requires treatment with cyclosporin A, a
strong T-cell-suppressive agent.17,18 Consequently,
immunotherapeutic approaches that exclusively rely on the induction
and/or redirection of cytotoxic T-cell responses are of limited
value in such clinical situations. This is reflected by preliminary
results of a clinical trial with autologous neuroblastoma cells
transduced with the IL-2 gene that resulted in a relatively low
response rate with one complete and one partial response among 14 stage
4 patients.16 Therefore, alternative approaches may be
preferable, including the use of a greater and possibly more effective
variety of immune cells, including T cells and NK cells.
We recently showed the feasibility of such an alternative approach to
cancer immunotherapy that combines the effective concentration of
cytokines in the tumor microenvironment with a technically simple
modus operandi.19 Thus, a recombinant fusion
protein consisting of tumor-specific antiganglioside GD2
antibody and IL-2 specifically induced a CD8+ T-cell
response, which was followed by a long-lasting, protective immunity in
a syngeneic mouse model of B78 melanoma cells that were transduced with
specific transferases to express high levels of the disialoganglioside
GD2.20-22 The ongoing clinical efforts in
combining IL-2 with anti-GD2 immunotherapy of neuroblastoma provided the rationale for evaluating the efficacy of a recombinant anti-GD2-IL-2 fusion protein in a pathophysiologically
relevant, preclinical model of murine neuroblastoma with experimental
metastases to bone marrow and liver.
Here, we extend our preclinical findings on the ch14.18-IL-2 fusion
protein in a novel, GD2-positive murine neuroblastoma model
in immunocompetent A/J mice featuring experimental metastases to
various distant organ sites, including bone marrow, which are pathophysiologically highly relevant for human neuroblastoma. We show
for the first time that the ch14.18-IL-2 fusion protein can
effectively eradicate established bone marrow and liver metastases more
efficiently than equivalent mixtures of antibody and recombinant human
(rh) IL-2. Furthermore, we show that the effector mechanism involved is
exclusively dependent on NK cells. These data support the hypothesis
that an effective concentration of cytokines in the tumor
microenvironment stimulates NK cells sufficiently to elicit strong
antitumor responses in vivo. Therefore, the future clinical application
of this type of therapy seems warranted, particularly after high-dose
chemotherapy and peripheral blood stem cell rescue, because the NK-cell
system is not deficient in this adjuvant setting.23
| |
MATERIALS AND METHODS |
Mice
Syngeneic female A/J and C.B-17 severe combined immunodeficiency (SCID)
or C.B-17 SCID/BEIGE mice were obtained at 8 weeks of age from Jackson
Laboratory (Bar Harbor, ME) and Taconic Farms (Germantown, NY),
respectively. They were housed in the pathogen-free mouse colony at our
institution in groups of four mice each. Mice were fed ad libitum on
standard mouse laboratory chow. Animal experiments were performed
according to the National Institutes of Health Guide for the Care and
Use of Laboratory Animals.
Cells
The murine NXS2 hybrid neuroblastoma cell line was created by fusion of
the GD2-negative C1300 murine neuroblastoma cell line (A/J
background) with murine dorsal root ganglional cells from C57BL/6J
mice,24 followed by fluorescence and magnetic-activated cell sorting for high GD2 expression, as previously
described.25 This hybrid cell line was shown to be major
histocompatibility complex (MHC) class I syngeneic to A/J mice, as
shown by its H2Kk-positive/H2Kb-negative
phenotype.25 GD2-positive NXS2 hybrid
neuroblastoma and NK-resistant P815 cells were maintained in
Dulbecco's minimal essential medium. GD2-negative
TBJ mouse neuroblastoma and NK-sensitive YAC1 cells were
cultured in RPMI 1640 at 5% CO2 and 37°C. All media
were supplemented with 2 mmol/L glutamine and 10% fetal calf serum
(FCS).
Antibody and Antibody-IL-2 Fusion Protein
Mouse-human chimeric antibody ch14.18, directed against
disialoganglioside GD2, was constructed by joining the cDNA
for the variable region of the murine antibody with the constant
regions of the 1 heavy chain and the  light chain, as described
previously.26 The ch14.18-IL-2 fusion protein was
constructed by fusion of a synthetic sequence coding for human IL-2 to
the carboxyl terminal of the human C1 gene, as
described.27 The fused gene was introduced into the vector
pdHL2, which encodes the dihydrofolate reductase gene. The expression
plasmid was introduced into Sp2/0-Ag14 cells and selected in the
presence of increasing concentrations of methotrexate (100 nmol/L to 5 µmol/L). The fusion protein was purified on Protein A Sepharose, and
its specific IL-2 activity was determined in bioassays, as described
previously.19,28 ch14.18-IL-2 (1 µg) was found to be
equivalent to 3,000 IU rhIL-2.19,28
Experimental Bone Marrow and Liver Metastases
NXS2 cells were harvested by trypsinization and washed three times by
centrifugation. Tumor cells were used for induction of metastases only
if their cell viability exceeded 95%, as determined by trypan blue
staining. Experimental metastases were induced by tail vein injection
of either 1 × 106 or 5 × 104 NXS2
cells, respectively, and mice were sacrificed for evaluation after 21 or 26 to 28 days. The number of metastatic liver foci, the percentage
of metastatic liver surface, and the liver weight was determined on
fresh specimens. For evaluation of bone marrow metastases, the bone
cavities of both femurs and tibiae of each animal were flushed with 3 mL phosphate-buffered saline (PBS; pH 7.4). The cell pellet was used
for total RNA isolation and subsequent reverse-transcription polymerase
chain reaction (RT-PCR) for the detection of tyrosine hydroxylase.
Established liver and bone marrow metastases were detected by
sequential analysis of liver and bone marrow in a group of eight mice 5 days after intravenous injection of 5 × 104 NXS2
cells. Livers were divided in half. One half was fixed in 10% buffered
formalin followed by paraffin embedding. Sections of three different
levels were stained with hematoxylin eosin (HE) and examined
microscopically for micrometastases. The second half of each liver was
homogenized and lysed in 2 mL RLT buffer, RNeasy (Qiagen, Chatsworth,
CA), for subsequent RNA isolation and RT-PCR for tyrosine hydroxylase.
RNA Isolation, RT, and PCR Amplification
Total cellular RNA was isolated by using a commercially available
silica gel membrane binding procedure, RNeasy (Qiagen), followed by the
synthesis of cDNA with 1 µg RNA in the presence of moloney murine
leukemia virus reverse transcriptase, SuperScriptII (GIBCO-BRL, Grand
Island, NY), according to manufacturer's guidelines. A denatured cDNA
equivalent of 100 ng was used in a 25 µL PCR reaction mixture, which
contained 20 mmol/L Tris-HCl (pH 8.4), 50 mmol/L KCl, 0.2 mmol/L
deoxynucleotide triphosphate, 2.5 U of Taq DNA polymerase (Grand
Island, NY), and 0.5 µmol/L sense and anti-sense oligonucleotide
primers for amplification of mouse tyrosine hydroxylase. For the
detection of tyrosine hydroxylase, the PCR was adjusted at low and high
sensitivity, as described previously. Briefly, for low sensitivity,
amplification was done with sense 5 TCT CAC TTC TTG AAG GAA CG
3 and anti-sense 5 CCC CAT TCT GTT TAC ACA GC 3
for 36 cycles (15 seconds at 96°C, 30 seconds at
63°C, 90 seconds at 72°C) leading to a 325-bp fragment designated TH1. High sensitivity was achieved by nested amplification of 1.5 µL TH1 after 20 cycles using sense 5 AGT ACA TCC GTC
ATG CCT CC 3 and anti-sense 5 GAG ATG CAA GTC CAA TGT CC
3 for 30 cycles to create a 132-bp fragment designated TH2. TH1
and TH2 PCR fragments were analyzed by polyacrylamide gel
electrophoresis. The sensitivity of TH1 and TH2 tyrosine hydroxylase
RT-PCR was established in reconstituion experiments at detection
thresholds of one NXS2 cell in 102 or 105 bone
marrow cells, respectively.25 If amplification revealed neither TH1 nor TH2 signals, the cDNA integrity was tested by amplification of glycerol-aldehyde-phosphate-dehydrogenase (GAPDH) with
sense 5 CAT TGA CCT CAA CTA CAT GG 3 and anti-sense
5 CAC ACC CAT CAC AAA CAT GG 3 leading to a 295-bp
fragment. The specificity of all fragments was verified by molecular
sequencing. According to high- and low-sensitivity tyrosine hydroxylase
RT-PCR results, bone marrow metastasis was designated as stage 0 with no PCR signal, stage 1 with an exclusive TH2 signal, and stage 2 in the
presence of both TH1 and TH2 signals.
Cytotoxicity Assays
Effector cells were prepared from mouse spleen cells by hypotonic lysis
of red blood cells with ACK lysis buffer (GIBCO-BRL, Gaithersburg, MD)
and either used for the cytotoxicity assay or for subsequent separation
into subpopulations. Enriched NK-cell populations and pure
CD8+ effector cells were prepared by magnetic activated
cell sorting, Mini MACS (Miltenyi Biotec, Auburn, CA). Briefly, mouse
splenocytes were incubated with either anti-mouse CD8 microbeads
(Miltenyi Biotec) or biotinylated pan NK-cell antibody DX5 (Pharmingen, San Diego, CA) and followed by labeling with streptavidin microbeads (Miltenyi Biotec). The magnetic-activated cell sorting was performed according to manufacturer's guidelines. The purity of the cell fractions was determined by fluorescence-activated cell sorter (FACS)
analysis. Target cells were incubated in the presence of 0.5 µCi
Na251CrO4 (Amersham, Cleveland, OH)
for 2 hours at 37°C, washed three times, and seeded in a
flat-bottom 96-well plate at a density of 5,000 cells per well.
Effector cells were added at various effector-to-target cell ratios in
a final volume of 200 µL per well and incubated for 4 or 18 hours.
Total release was induced with 5 µL sodium dodecyl sulfate
(SDS;10%). The supernatant was collected from each well for
determination of 51Cr release. The percentage of target
cell lysis was calculated as follows:
The
results were expressed as mean value ± standard deviation of at
least three experiments.
Immunohistology
Frozen sections of livers were fixed in cold acetone for 10 minutes,
followed by removal of endogeneous peroxidase with 0.03% H2O2 for 30 minutes at room temperature.
Endogenous biotin was removed from liver tissues by incubation with
0.1% avidin for 10 minutes at room temperature, followed by buffer
rinses. Excess avidin was neutralized by treatment with low
concentration biotin (0.01%) for 10 minutes at room temperature.
Nonspecific binding was blocked with 10% species-specific serum in 1%
bovine serum albumin (BSA)/PBS. Biotinylated anti-mouse CD45 MoAb
(Pharmingen), biotinylated anti-mouse CD8, and NK-cell-specific rabbit
anti-asialo GM1 antiserum (WAKO, Richmond, VA) were diluted to 10 µg/mL in 10% goat serum (1% BSA/PBS, pH 7.4) and overlaid onto
serial sections. Slides were incubated in a humid chamber for 30 minutes followed by the application of a biotinylated goat anti-rabbit
antibody onto slides with anti-asialo GM1 antiserum preincubation for
10 minutes. Streptavidin-labeled alkaline phosphatase was incubated for
10 minutes followed by substrate development using the
VectorR Red substrate kit (Vector Laboratories, Burlingame,
CA) and hematoxylin nuclear counterstain. All incubations were followed
by three wash steps with 0.05 mol/L Tris with 150 mmol/L NaCl, pH 8.0 on sections that received the alkaline phosphatase-conjugated
streptavidin and PBS (pH 7.4) on slides that received the
peroxidase-conjugated streptavidin.
Statistics
The statistical significance of differential findings between
experimental groups of animals was determined by two-tailed Student's
t-test. The nonparametric Wilcoxon test was used to determine
the statistical significance of metastatic scores. Findings were
regarded as significant if two-tailed P values were <.01.
| |
RESULTS |
Effector Mechanisms of the Immune Response Induced by the
ch14.18-IL-2 Fusion Protein
In Vivo.
We previously reported the complete growth suppression of experimental
liver and bone marrow metastases of neuroblastoma by treatment with
ch14.18-IL-2 fusion protein, an effect that could not be achieved with
a mixture of ch14.18 and rhIL-2 at equivalent concentrations.25 Again, only mice treated with the fusion
protein (10 µg, ×6) revealed no signs of experimental
metastases to either bone marrow or liver, as determined by RT-PCR of
tyrosine hydroxylase or liver weight and count of macroscopic tumor
foci, respectively (Table 1). In addition,
we proved the specificity of the ch14.18-IL-2 fusion protein therapy,
because its complete treatment effect was abrogated against
GD2 target antigen-negative TBJ mouse neuroblastoma cells
(Table 1).
View this table:
[in this window]
[in a new window]
|
Table 1.
Therapeutic Effect of Anti-GD2
Antibody-IL-2 Fusion Protein on Experimental Liver and Bone Marrow
Metastases in Immunocompetent and Immune-Deficient Mice
|
|
The effector mechanisms involved in the treatment effect were
delineated by using immunodeficient (Table 1) and in vivo-depleted immunocompetent mice (Table 2). In a first
set of experiments, we used T- and B-cell-deficient C.B17 SCID mice
and C.B17 SCID/BEIGE mice deficient in both T and B cells, as well as
NK cells. The ch14.18-IL-2 fusion protein was completely effective in
the T- and B-cell-deficient strain. In contrast, the additional
absence of NK cells in the SCID/BEIGE mouse completely abrogated the
antitumor effect of the fusion protein, implying a mechanism mediated
by NK cells. Two additional lines of evidence further established proof
for an NK-cell-mediated mechanism evoked by the ch14.18-IL-2 fusion
protein. First, the treatment effect was fully restored when SCID/BEIGE
mice were reconstituted with NK cells, generated in vitro by incubation
of splenocytes from naïve A/J mice with 1,000 IU/mL rhIL-2
(Table 2). Second, depletion of NK cells in A/J mice by anti-asialo GM1
antiserum abrogated the effect of the ch14.18-IL-2 fusion protein,
whereas depletion of CD8+ T cells by anti-CD8 antibody had
no effect on the efficacy of the fusion protein treatment (Table 2).
View this table:
[in this window]
[in a new window]
|
Table 2.
Therapeutic Effect of Anti-GD2
Antibody-IL-2 Fusion Protein on Experimental Liver Metastasis of NXS2
Cells in Immunocompetent and In Vivo-Depleted A/J Mice
|
|
In Vitro.
An NK-cell mechanism mediated by the ch14.18-IL-2 fusion protein was
also shown in vitro by a strong stimulation of NK-cell-mediated lysis
against YAC1 and NXS2 target cells after treatment with ch14.18-IL-2
fusion protein (Fig 1). In
this case, tumor-bearing mice were treated with daily intravenous
injections (×6) of either 10 µg ch14.18-IL-2 fusion protein, a
mixture of 10 µg ch14.18 antibody plus an equivalent amount of rhIL-2
(30,000 IU), or PBS (pH 7.4). When these animals were killed 1 day
after completion of the treatment, only splenocytes of mice treated
with ch14.18-IL-2 fusion protein showed strong NK activity in a 4-hour
chromium release assay (Fig 1A). In contrast, splenocytes from mice
treated with PBS or the antibody/IL-2 mixture mediated marginal lysis of NK-sensitive YAC1 cells only at high effector to target cell ratios.
Lysis of NK-resistant P815 murine mastocytoma cells by splenocytes of
mice that received the ch14-18-IL-2 fusion protein therapy is shown as
a control (Fig 1A). This fusion protein-induced stimulation was only
inhibited after in vivo depletion of NK cells with anti-asialo GM1
antiserum (Fig 1B). No inhibition of lysis was observed after depletion
of CD8+ T cells with anti-CD8 antibody (Fig 1B), which is
in agreement with the demonstrated therapeutic effect of the
ch14.18-IL-2 fusion protein in vivo (Table 2). After successful tumor
therapy with fusion protein, splenocytes from A/J mice produced the
strongest lysis of NXS2 cells in the presence of 10 µg/mL
ch14.18-IL-2. However, this lysis was less effective in the presence
of either equivalent mixtures of ch14.18 antibody and IL-2 or ch14.18
and IL-2 alone (Fig 1C). After magnetic-activated cell sorting of splenocytes from fusion protein-treated mice with T-cell-specific anti-CD8 or NK-cell-specific DX5 antibodies, cytotoxic activity against NXS2 cells was only evident in the NK-cell fraction; only background lysis was shown with pure CD8+ T cells,
suggesting a T-cell-independent cytotoxic activity.
View larger version (23K):
[in this window]
[in a new window]
View larger version (25K):
[in this window]
[in a new window]
View larger version (14K):
[in this window]
[in a new window]
| Fig 1.
Cytotoxic activity of splenocytes from
tumor-bearing A/J mice after ch14.18-IL-2 fusion protein therapy. (A)
Experimental metastasis was induced by intravenous injection of 1 × 106 NXS2 cells followed by six daily intravenous
administrations of either 10 µg ch14.18-IL-2, an equivalent mixture
of ch14.18 antibody and rhIL-2, or PBS. Twenty-four hours after
completion of the treatment, the cytotoxic activity of splenocytes was
tested in a 4-hour chromium release assay against YAC1 (closed symbols) and P815 (open symbols) target cells. (B) Splenocytes of
immunocompetent (closed symbols) and in vivo-depleted mice (open
symbols) were tested for lysis against YAC1 cells in a 4-hour chromium
release assay 24 hours after completion of the treatment with PBS or 10 µg ch14.18-IL-2 fusion protein (daily, ×6, starting 24 hours after induction of experimental metastases). Immunodepletion was started at
day  3 and 1 before tumor cell inoculation followed by a
once-weekly schedule with intraperitoneal injection of either 350 µg
anti-CD8 antibody or 100 µL anti-asialo GM1 antiserum, respectively.
(C) Splenocytes of tumor-bearing mice treated with 10 µg
ch14.18-IL-2 fusion protein (daily, ×6, starting 24 hours after
induction of experimental metastases) were tested for lysis of NXS2
target cells in a 4-hour chromium release assay. Splenocytes were used at an effector-to-target-cell ratio of 100:1 in the presence of (a)
PBS; (b) 30,000 IU/mL rhIL-2; (c) 10 µg/mL ch14.18; (d) 30,000 IU/mL
rhIL-2+10 µg/mL ch14.18; and (e) 10 µg/mL ch14.18-IL-2. The
lysis of NK-cell-enriched (f) and pure CD8+ T-cell (g)
subfractions was compared with that of whole splenocytes (e) in the
presence of 10 µg/mL ch14.18-IL-2.
|
|
Effect of ch14.18-IL-2 Fusion Protein Treatment on Established
Bone Marrow and Liver Metastases
To document established bone marrow and liver metastasis, specimens of
both organ systems were collected from eight mice each 5 days after
injection of 5 × 104 NXS2 cells. The presence of
tumor cells was detected by tyrosine hydroxylase RT-PCR and histology.
All mice had established liver metastases at that time, as shown by the
presence of a TH2 signal, which indicates detection of liver metastases
at a sensitivity of one tumor cell in 106 hepatocytes
(Fig 2C). Microscopic examination of such
livers revealed tumor foci with an average size of 5 to 15 cells, as depicted in Fig 2D. This same analysis of bone marrow on day 5 after
tumor cell inoculation indicated established metastases by the presence
of a TH2 signal in at least 50% of mice (Fig 2A). Amplification of
murine GAPDH was used as a control template for bone marrow samples
without a TH2 signal (Fig 2A). It should be noted that the sensitivity
of detecting NXS2 cells in naïve bone marrow by tyrosine
hydroxylase nested RT-PCR is only one tumor cell in 105
bone marrow cells, as previously described.25 However,
histological examination of bone marrow specimens also revealed 5 to 15 tumor cell foci, as depicted in Fig 2B. Treatment of such mice with established liver and bone marrow metastases by six daily injections of
40 µg ch14.18-IL-2 fusion protein resulted in complete eradication of bone marrow and liver disease (Table 3).
View larger version (73K):
[in this window]
[in a new window]
| Fig 2.
Demonstration of established bone marrow and liver
metastasis after intravenous injection of 5 × 104 NXS2
cells by histology and tyrosine hydroxylase RT-PCR. Mice (n = 8) were
killed 5 days after intravenous injection with 5 × 104
NXS2 cells. Bone marrow (A and B) and liver (C and D) specimens were
analyzed by tyrosine hydroxylase- nested RT-PCR (A and C, top) and
histology (B and D). The presence of a TH2 signal indicates NXS2
infiltration into bone marrow (A) or liver (C). GAPDH was amplified
with probes lacking a TH2 signal to prove cDNA integrity (A, bottom).
A sensitivity of one tumor cell in 106
hepatocytes was established with tyrosine hydroxylase-nested RT-PCR of
NXS2 cells in liver tissue was established with reciprocal tumor to
liver cell ratios of 1:104 to 1:107 (C, lanes 9 to 12). Paraffin-embedded sections of bone marrow and liver specimen
were stained with hematoxylin/eosin. Arrows indicate focal tumor cell
infiltrates photographed at 1,000 × magnification (oil immersion).
|
|
View this table:
[in this window]
[in a new window]
|
Table 3.
Therapeutic Effect of Anti-GD2 Antibody IL-2
Fusion Protein on Established Experimental Metastasis of NXS2 Cells in
C.B-17 SCID Mice
|
|
Mechanism of the Antitumor Response Induced by the ch14.18-IL-2
Fusion Protein Against Established NXS2 Neuroblastoma Metastases
A reduction in dosage from 40 µg (×6) to 10 µg ch14.18-IL-2
(×5) still resulted in a significant reduction of liver weights in fusion protein treated animals with established liver metastases (P < .001; Table 4). Histological
examination of livers from such mice revealed an infiltrate of
polymorphonuclear cells as well as mononuclear cells
(Fig 3C). This is in contrast to livers from mice that showed no inflammatory infiltrates when treated with
either PBS (pH 7.4) or a mixture of 10 µg ch14.18 antibody plus an
equivalent amount of rhIL-2 (30,000 IU; Fig 3A and B). Immunophenotyping of such cellular infiltrates revealed strong staining
with anti-asialo GM1 antiserum, a well-established marker for mouse NK
cells (Fig 3E). In contrast, only background staining was observed in
adjacent sections with monoclonal anti-CD8 antibody, a marker for
cytotoxic T cells (Fig 3F). Staining with anti-mouse CD45 antibody,
recognizing a common leukocyte antigen, is shown as a control (Fig 3D).
None or only very weak staining was observed with monoclonal anti-CD4
antibody (data not shown). The lack of a CD8+
T-cell-mediated systemic immune response was shown when NXS2 tumor
challenge of A/J mice, previously successfully treated with ch14.18-IL-2 fusion protein, did not result in a delay of subcutaneous tumor growth (Table 4).
View this table:
[in this window]
[in a new window]
|
Table 4.
Effect of NK Cell Activation on Anti-GD2
Antibody IL-2 Fusion Protein Therapy of Established Experimental
Neuroblastoma Metastases
|
|
View larger version (180K):
[in this window]
[in a new window]
| Fig 3.
Histological and immunohistochemical analyses of
tumor-bearing mice after fusion protein therapy. Five days after
intravenous inoculation with 5 × 104 NXS2 cells, mice
with established liver metastases were treated by daily (×5)
intravenous injections of either 10 µg ch14.18-IL-2 fusion protein,
an equivalent mixture of 10 µg ch14.18 and 30,000 IU rhIL-2, or PBS
(pH 7.4). Twenty-four hours after completion of treatment,
paraffin-embedded sections of livers from either PBS (A), IL-2/antibody
mixture (B), and fusion protein (C) treated animals were stained with
hematoxylin/eosin. Tumor foci or infiltrates are depicted at a
magnification of 1,000×. Arrows delineate tumor foci (A and B) or
inflammatory cells (C). Frozen liver sections of mice treated with
fusion protein were stained with monoclonal anti-leukocyte CD45
antibody (D), anti-asialo GM1 antiserum (E), and monoclonal anti-CD8
antibody (F). Red cells indicate positive staining for each marker.
|
|
Additional activation of NK cells with
polyriboinosinic:polyribocytidylic acid (poly IC; 100 µg
intraperitoneally, ×3, days 4 to 6) or recombinant mouse
interferon- (rmIFN; 300,000 IU subcutaneously/6 days,
ALZET micro-osmotic pump [Alza Corp, Palo Alto, CA])
resulted in an increased antitumor response if applied in combination
with the ch14.18-IL-2 fusion protein (Table 4). In fact, the
combination with rmIFN- led to a complete eradication of liver and
bone marrow metastases (Table 4). Splenocytes from these animals,
collected 24 hours after completion of treatment, produced the highest
in vitro cytotoxic effect against NXS2 target cells. In comparison,
splenocytes from mice treated with either the fusion protein or
rmIFN- alone were far less effective
(Fig 4). The question was addressed of
whether the mIFN--induced increase in expression of MHC class I
antigens is followed by a switch from NK-cell to T-cell effector
activity. In fact, FACS analyses showed a sevenfold increase of
H2Kk expression after incubation of NXS2 cells with 100 IU
mIFN- for 48 hours in vitro. Furthermore, the subcutaneous
application of 300,000 IU mIFN- by way of micro-osmotic pump over 6 days led to a threefold increase in H2Kk expression in
subcutaneous tumors that were induced by subcutaneous injection of 5 × 106 NXS2 cells 3 days before mIFN- application
(data not shown). However, in spite of this increase in MHC class I
antigens, splenocytes from mice with established metastases that were
successfully treated with a combination therapy of ch14.18-IL-2 fusion
protein and mIFN- did not kill NXS2 target cells in an MHC class
I-restricted fashion (Fig 4). On the contrary, the addition of
anti-MHC class I antibodies to the assay actually increased the
cytolytic effect of splenocytes obtained from animals 24 hours after
the completion of the ch14.18-IL-2 and mIFN- combination treatment
(Fig 4). This increase cannot be attributed to antibody-dependent
cellular cytotoxicity mediated by the addition of H2Kk MHC
class I antibodies, because lysis of NXS2 cells with splenocytes from
PBS-treated control animals was not increased in the presence of these
antibodies (data not shown). These data suggest an inhibitory effect of
MHC class I antigens in this system, which is, incidentally, a typical
feature of NK-cell-mediated responses.
View larger version (10K):
[in this window]
[in a new window]
| Fig 4.
In vitro lysis of NXS2 neuroblastoma cells by splenocytes
from tumor-bearing A/J mice after treatment with fusion protein. Five
days after intravenous inoculation with 5 × 104 NXS2
cells, mice with established liver metastases were treated by daily
(×5) intravenous injections with either 10 µg ch14.18-IL-2 fusion
protein or PBS (pH 7.4), in the presence or absence of additional
rmIFN- (300,000 IU/6 days subcutaneous via ALZET osmotic pump).
Cytotoxic activity was determined 24 hours after completion of
treatment in an 18-hour chromium release assay against NXS2 target
cells using splenocytes at an effector-to-target-cell ratio of 100:1.
Splenocytes were obtained from mice previously treated with either PBS
(a), rmIFN- (b), fusion protein (c), fusion protein + rmIFN-
(d), or additional anti-H2Kk antibody (25 µg/mL) in vitro
(e).
|
|
| |
DISCUSSION |
The induction of an effective cellular immune response against
syngeneic tumors by a local increase of inflammatory cytokines in the
tumor microenvironment is a promising approach in hematology/oncology. In contrast to patient-specific ex vivo transduction of an
individual's tumor cells or fibroblasts by cytokine genes, our
approach is an attempt to induce a cellular antitumor immune response
by using a fusion protein to direct cytokines to the tumor
microenvironment. Such recombinant antibody-cytokine fusion proteins
combine the unique targeting ability of antibodies with the
inflammatory activity of cytokines and can be applied by a simple
modus operandi in a patient-independent manner.
Here, we show the superiority of a recombinant anti-GD2
ch14.18-IL-2 fusion protein over a mixture of equivalent amounts of this cytokine with ch14.18 antibody in effectively eradicating established experimental neuroblastoma metastases to bone marrow and
liver. This effect was achieved by NK cells in a novel, immune competent syngeneic model for murine neuroblastoma that naturally expresses GD2. This tumor model has many pathophysiological
similarities with human neuroblastoma and expresses the tumor marker
tyrosine hydroxylase. In fact, two thirds of neuroblastoma patients
initially present with bone marrow metastases.29 Most of
these patients suffer from minimal residual disease to the bone marrow
after conventional therapy protocols and high-dose chemotherapy
followed by peripheral stem cell rescue. This was substantiated by
highly sensitive detection systems such as anti-GD2
immunohistochemistry or tyrosine hydroxylase RT-PCR.30-33
Here, we present the first evidence for a mechanism by which the
ch14.18-IL-2 fusion protein induces NK-cell-mediated suppression of
tumor growth and eradication of established bone marrow and liver
metastases in a syngeneic model for murine neuroblastoma. This is in
contrast to a previously described mechanism by which this same fusion
protein induced a T-cell-mediated immune response, independent of NK
cells, in a syngeneic model for murine melanoma.20-22 To
assess whether low expression of MHC class I antigens might favor NK
cells over T cells in this neuroblastoma tumor model, the expression of
these antigens was increased by the addition of mIFN-. Although
mIFN- strongly upregulated MHC class I expression in vitro and in
vivo, splenocytes from mice successfully treated with a combination
therapy of ch14.18-IL-2 fusion protein and mIFN- were incapable of
MHC class I-restricted killing of NXS2 target cells. Thus, a switch
from an NK- to a T-cell-mediated mechanism did not occur. In fact,
quite the opposite was observed as the addition of anti-MHC class I
antibodies increased the cytotolytic effect of splenocytes obtained
from mice 24 hours after completion of the ch14.18-IL-2/mIFN-
combination treatment. This is a pattern for NK-cell-mediated killing
of tumor cells, beause NK-cell activity can be downregulated by
properly stimulated lectin type-C inhibitory NK-cell receptors of the
LY49 family that are specific for MHC class I
molecules.34,35 In our experiments this downregulation of
NK-cell activity was counteracted by the addition of MHC class I
blocking antibodies. Therefore, the increase achieved in antitumor activity of the ch14.18-IL-2 fusion protein by the further application of mIFN- in vivo can be attributed to an additional stimulation of
NK cells. This contention is supported by the finding that splenocytes
from mice treated with the ch14.18-IL-2/mIFN- combination therapy
achieved the highest lysis of YAC1 cells, as compared with mIFN- and
ch14.18-IL-2 controls (data not shown).
The exclusive response by NK cells in our neuroblastoma model was
underlined by the effect of subcutaneous tumor cell challenge on fusion
protein-treated mice. Specifically, when such mice were challenged by
subcutaneous injection of NXS2 cells 3 days after completion of the
treatment, they developed subcutaneous tumors similar to untreated mice
or mice receiving the equivalent mixture of antibody and IL-2 (Table
4). The absence of a T-cell-mediated immune response in this
particular model and its concomitant replacement by NK cells might be
caused by factors secreted by the tumor cells that suppress T cells but
stimulate NK cells. In fact, we observed that NXS2 cells produce
transforming growth factor- 1 (TGF-1) and IL-10 in vitro and in
vivo (data not shown), both immunomodulators associated with T-cell
anergy.36-41 However, IL-10 is a factor that was also found
to stimulate NK cells, because they showed an increase in
[3H]thymidine uptake and killing of NK-sensitive target
cells after incubation with IL-10.42 However, it was also
shown that IL-10 can inhibit tumor metastasis by an NK-cell-dependent
mechanism.43 Consequently, it is possible that the presence
of TGF-1 and IL-10 in our model may favor NK-cell-dependent
antitumor mechanisms by causing NK-cell stimulation and concomitant
T-cell anergy.
The availability of effective adjuvant treatment for neuroblastoma in
the postchemotherapy and transplant phase remains as a major challenge
in pediatric hematology/oncology. In this regard, the effectiveness of
the ch14.18-IL-2 fusion protein is striking, especially in view of the
effector mechanism involved. Thus, we could clearly show in vivo and in
vitro that the ch14.18-IL-2 fusion protein stimulates a cellular
antitumor response exclusively mediated by NK cells. It is of
considerable interest that NK cells, in contrast to T cells, were not
found to be deficient in patients with solid and hematological
malignancies, even after high-dose chemotherapy followed by autologous
peripheral blood stem cell transplantation.23 This finding
suggests that neuroblastoma patients who are deficient in T cells
following high-dose chemotherapy still have NK effector cells that, if
properly stimulated, can effectively elicit an antitumor response. When
tumor growth and progression of patients can be held to a minimum, they
may eventually recover their T-cell-dependent immune system. Once this
is achieved, T-cell-dependent effector mechanisms could become more
effective for tumor cell killing. However, because most human
neuroblastoma cells or cell lines show a low expression of MHC class I
molecules,44,45 few, if any, effective T-cell-dependent
antitumor responses, followed by a protective immunity with a T-cell
memory, were observed thus far in neuroblastoma. Because stimulated NK
cells proved to be very effective in our animal model in the absence of
a memory immune response, multiple treatments with the ch14.18-IL-2
fusion protein may well be required to achieve optimal antitumor
responses in neuroblastoma patients.
In summary, we show here that NK cells stimulated by the ch14.18-IL-2
fusion protein can effectively suppress tumor dissemination and growth
and effectively eradicate established bone marrow metastasis in a
syngeneic model of neuroblastoma in A/J mice. The mechanism responsible
for this antitumor effect proved to be exclusively dependent on NK
cells. Taken together, our preclinical data described here suggest that
the application of the ch14.18-IL-2 fusion protein in an adjuvant
setting may lead to further improvement in the treatment of
neuroblastoma patients with minimal residual disease.
| |
FOOTNOTES |
Submitted September 8, 1997;
accepted October 20, 1997.
Supported by the National Institutes of Health Outstanding
Investigator's Award Grant No. CA-42508 (R.A.R.). H.N.L. was supported by a training grant of the Deutsche Forschungsgemeinschaft.
Address reprint requests to Ralph A. Reisfeld, PhD, The Scripps
Research Institute, Department of Immunology, 10550 N Torrey Pines Rd,
IMM13, La Jolla, CA, 92037.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Carrie Dolman for her excellent technical assistance and also
express our appreciation to Lynne Kottel for the preparation of this
manuscript. We extend special thanks to Petra Kleindienst for her
dedicated help with FACS and RT-PCR analyses.
| |
REFERENCES |
1. Niethammer D, Handgretinger R: Clinical strategies for the
treatment of neuroblastoma. Eur J Cancer 31A:568, 1995
2. Handgretinger R, Anderson K, Lang P, Dopfer R, Klingebiel T,
Schrappe M, Reuland P, Gillies SD, Reisfeld RA, Neithammer D: A phase I
study of human/mouse chimeric anti-ganglioside GD2 antibody
ch14.18 in patients with neuroblastoma. Eur J Cancer 31A:261, 1995
3.
Handgretinger R,
Baader P,
Dopfer R,
Klingebiel T,
Reuland P,
Treuner J,
Reisfeld RA,
Niethammer D:
A phase I study of neuroblastoma with the anti-ganglioside GD2 antibody 14.G2a.
Cancer Immunol Immunother
35:199,
1992[Medline]
[Order article via Infotrieve]
4.
Cheung NK,
Cheung IY,
Canete A,
Yeh SJ,
Kushner B,
Bonilla MA,
Heller G,
Larson SM:
Antibody response to murine anti-GD2 monoclonal antibodies: Correlation with patient survival.
Cancer Res
54:2228,
1994[Abstract/Free Full Text]
5.
Uttenreuther-Fischer MM,
Huang CS,
Reisfeld RA,
Yu AL:
Pharmacokinetics of anti-ganglioside GD2 mAb 14G2a in a phase I trial in pediatric cancer patients.
Cancer Immunol Immunother
41:29,
1995[Medline]
[Order article via Infotrieve]
6.
Riethmueller G,
Schneider-Gaedicke E,
Schlimok G,
Schmiegel W,
Raab R,
Hoffken K,
Gruber R,
Pichlmaier H,
Hirche H,
Pichelmayr R,
Buggisch P,
Witte J:
Randomised trial of monoclonal antibody for adjuvant therapy of resected DukesC colorectal carcinoma (The German Cancer Aid 17-1A Study group).
Lancet
94:1177,
1994
7.
Honsik CJ,
Jung G,
Reisfeld RA:
Lymphokine-activated killer cells targeted by monoclonal antibodies to the disialogangliosides GD2 and GD3 specifically lyse human tumor cells of neuroectodermal origin.
Proc Natl Acad Sci USA
83:7893,
1986[Abstract/Free Full Text]
8.
Shiloni E,
Eisenthal A,
Sachs D,
Rosenberg SA:
Antibody-dependent cellular cytotoxicity mediated by murine lymphocytes activated in recombinant interleukin 2.
J Immunol
138:1992,
1987[Abstract]
9.
Grimm EA,
Mazumder A,
Zhang HZ,
Rosenberg SA:
Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.
J Exp Med
155:1823,
1982[Abstract/Free Full Text]
10.
Grimm EA,
Ramsey KM,
Mazumder A,
Wilson DJ,
Djeu JY,
Rosenberg SA:
Lymphokine-activated killer cell phenomenon. II. Precursor phenotype is serologically distinct from peripheral T lymphocytes, memory cytotoxic thymus-derived lymphocytes, and natural killer cells.
J Exp Med
157:884,
1983[Abstract/Free Full Text]
11.
Hank JA,
Robinson RR,
Surfus J,
Mueller BM,
Reisfeld RA,
Cheung NK,
Sondel PM:
Augmentation of antibody dependent cell mediated cytotoxicity following in vivo therapy with recombinant interleukin-2.
Cancer Res
50:5234,
1990[Abstract/Free Full Text]
12.
Rosenberg SA,
Yannelli JR,
Yang JC,
Topalian SL,
Schwartzentruber DJ,
Weber JS,
Parkinson DR,
Seipp CA,
Einhorn JH,
White DE:
Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2.
J Natl Cancer Inst
86:1159,
1994[Abstract/Free Full Text]
13.
Rosenberg SA,
Lotze MT,
Yang JC,
Aebersold PM,
Linehan WM,
Seipp CA,
White DE:
Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients.
Ann Surg
210:474,
1989[Medline]
[Order article via Infotrieve]
14.
Favrot MC,
Floret D,
Negrier S,
Cochat P,
Bouffet E,
Zhou DC,
Franks CR,
Bijman T,
Brunat-Mentigny M,
Philip I,
Philip T:
Systemic interleukin-2 therapy in children with progressive neuroblastoma after high dose chemotherapy and bone marrow transplantation.
Bone Marrow Transplant
4:499,
1989[Medline]
[Order article via Infotrieve]
15.
Frost JD,
Hank JA,
Reaman GH,
Frierdich SRN,
Seeger RC,
Gan J,
Anderson PM,
Ettinger LJ,
Cairo MS,
Blazar BR,
Krailo MD,
Matthay KK,
Reisfeld RA,
Sondel PM:
Phase I/IB trial of murine monoclonal anti-GD2 antibody 14.G2a plus IL-2 in children with refractory neuroblastoma: A report of the children's cancer group.
Cancer
80:317,
1997[Medline]
[Order article via Infotrieve]
16.
Roth JA,
Cristiano RJ:
Gene therapy for cancer: What have we done and where are we going.
J Natl Cancer Inst
89:21,
1997[Abstract/Free Full Text]
17.
Schwinghammer TL,
Bloom EJ:
Pharmacologic prophylaxis of acute graft-versus-host disease after allogeneic marrow transplantation [review].
Clinical Pharmacy
12:736,
1993[Medline]
[Order article via Infotrieve]
18.
Parkman R:
Is chronic graft versus host disease an autoimmune disease? [review].
Curr Opin Immunol
5:800,
1993[Medline]
[Order article via Infotrieve]
19.
Sabzevari H,
Gillies SD,
Mueller BM,
Pancook JD,
Reisfeld RA:
A recombinant antibody-interleukin 2 fusion protein suppresses growth of hepatic human neuroblastoma metastases in severe combined immunodeficiency mice.
Proc Natl Acad Sci USA
91:9626,
1994[Abstract/Free Full Text]
20.
Becker JC,
Pancook JD,
Gillies SD,
Furukawa K,
Reisfeld RA:
T-cell-mediated eradication of murine metastatic melanoma induced by targeted interleukin 2 therapy.
J Exp Med
183:2361,
1996[Abstract/Free Full Text]
21.
Becker JC,
Varki NM,
Gillies SD,
Furukawa K,
Reisfeld RA:
An antibody-interleukin 2 fusion protein overcomes tumor heterogeneity by induction of a cellular immune response.
Proc Nat Acad Sci USA
93:7826,
1996[Abstract/Free Full Text]
22.
Becker JC,
Varki NM,
Gillies SD,
Furukawa K,
Reisfeld RA:
Long-lived and transferable tumor immunity in mice after targeted interleukin-2 therapy.
J Clin Invest
98:2801,
1997[Medline]
[Order article via Infotrieve]
23.
Koehne G,
Zeller W,
Stockschlaeder M,
Zander AR:
Phenotype of lymphocyte subsets after autologous peripheral blood stem cell transplantation.
Bone Marrow Transplant
19:149,
1997[Medline]
[Order article via Infotrieve]
24.
Greene LA,
Shain W,
Chalazonitis A,
Breakfield X,
Minna J,
Coon HG,
Nirenberg M:
Neuronal properties of hybrid neuroblastoma X sympathetic ganglion cells.
Proc Natl Acad Sci USA
72:4923,
1975[Abstract/Free Full Text]
25.
Lode HN,
Xiang R,
Varki NM,
Dolman CS,
Gillies SD,
Reisfeld RA:
Targeted interleukin-2 therapy of spontaneous neuroblastoma metastases to bone marrow.
J Natl Cancer Inst
89:1586,
1997[Abstract/Free Full Text]
26.
Gillies SD,
Lo KM,
Wesolowski J:
High-level expression of chimeric antibodies using adapted cDNA variable region casettes.
J Immunol Methods
125:191,
1989[Medline]
[Order article via Infotrieve]
27.
Gillies SD,
Reilly EB,
Lo KM,
Reisfeld RA:
Antibody-targeted interleukin 2 stimulates T cell killing of autologous tumor cells.
Proc Natl Acad Sci USA
89:1428,
1992[Abstract/Free Full Text]
28.
Hank JA,
Surfus JE,
Gan J,
Jaeger P,
Gillies SD,
Reisfeld RA,
Sondel PM:
Activation of human effector cells by a tumor reactive recombinant anti-ganglioside GD2 interleukin-2 fusion protein (ch14.18-IL-2).
Clin Cancer Res
2:1951,
1996[Abstract]
29.
Brodeur GM,
Pritchard J,
Berthold F,
Carlsen NL,
Castel V,
Castelberry RP,
De Bernardi B,
Evans AE,
Favrot M,
Hedborg F,
Kaneko M,
Kemshead J,
Lampert F,
Lee REJ,
Look AT,
Pearson ADJ,
Philip T,
Roald B,
Sawada T,
Seeger RC,
Tsuchida Y,
Voute PA:
Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment.
J Clin Oncol
11:1466,
1993[Abstract/Free Full Text]
30. Burchill SA, Bradbury FM, Selby P, Lewis IJ: Early clinical
evaluation of neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosine hydroxylase mRNA. Eur J Cancer 31A:553, 1995
31.
Miyajima Y,
Horibe K,
Fukuda M,
Matsumoto K,
Numata S,
Mori H,
Kato K:
Sequential detection of tumor cells in the peripheral blood and bone marrow of patients with stage IV neuroblastoma by the reverse transcription-polymerase chain reaction for tyrosine hydroxylase mRNA.
Cancer
77:1214,
1996[Medline]
[Order article via Infotrieve]
32.
Miyajima Y,
Kato K,
Numata S,
Kudo K,
Horibe K:
Detection of neuroblastoma cells in bone marrow and peripheral blood at diagnosis by the reverse transcriptase-polymerase chain reaction for tyrosine hydroxylase mRNA.
Cancer
75:2757,
1995[Medline]
[Order article via Infotrieve]
33.
Burchill SA,
Bradbury FM,
Smith B,
Lewis IJ,
Selby P:
Neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosine hydroxylase mRNA.
Int J Cancer
57:671,
1994[Medline]
[Order article via Infotrieve]
34.
Raulet DH,
Held W:
Natural killer cell receptors: The offs and ons of NK cell recognition.
Cell
82:697,
1995[Medline]
[Order article via Infotrieve]
35.
Raulet DH:
Recognition events that inhibit and activate natural killer cells.
Curr Opin Immunol
8:372,
1996[Medline]
[Order article via Infotrieve]
36.
Massague J:
The TGF-beta family of growth and differentiation factors.
Cell
49:437,
1987[Medline]
[Order article via Infotrieve]
37.
Roszman T,
Elliott L,
Brooks W:
Modulation of T cell function by gliomas.
Immunol Today
12:370,
1991[Medline]
[Order article via Infotrieve]
38.
Ruffini PA,
Rivoltini L,
Silvani A,
Boiardi A,
Parmiani G:
Factors, including transforming growth factor beta, released in the glioblastoma residual cavity, impair activity of adherent lymphokine-activated killer cells.
Cancer Immunol Immunother
36:409,
1993[Medline]
[Order article via Infotrieve]
39.
Schwartz RH:
Models of T cell anergy: Is there a common molecular mechanism?
J Exp Med
184:1,
1996[Free Full Text]
40.
Smith KA:
Lowest dose interleukin-2 immunotherapy.
Blood
81:1414,
1993[Free Full Text]
41.
Tigges MA,
Casey LS,
Koshland ME:
Mechanism of interleukin-2 signaling: Mediation of different outcomes by a single receptor and transduction pathway.
Science
243:781,
1989[Abstract/Free Full Text]
42.
Carson WE,
Lindemann MJ,
Baiocchi R,
Linett M,
Tan JC,
Chou CC,
Narula S,
Caligiuri MA:
The functional characterization of interleukin-10 receptor expression on human natural killer cells.
Blood
85:3577,
1995[Abstract/Free Full Text]
43.
Zheng LM,
Ojcius DM,
Garaud F,
Roth C,
Maxwell E,
Li Z,
Rong H,
Chen J,
Wang XY,
Catino JJ,
King I:
Interleukin-10 inhibits tumor metastasis through an NK cell-dependent mechanism.
J Exp Med
184:579,
1996[Abstract/Free Full Text]
44.
Murphy C,
Nikodem D,
Howcroft K,
Weissman JD,
Singer DS:
Active repression of major histocompatibility complex class I genes in a human neuroblastoma cell line.
J Biol Chem
271:30992,
1996[Abstract/Free Full Text]
45. van't Veer LJ, Beijersbergen RL, Bernards R: N-myc suppresses
major histocompatibility complex class I gene expression through
down-regulation of the p50 subunit of NF-kappa B. EMBO J 12:195, 1993
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
M. Gil, M. Bieniasz, A. Wierzbicki, B. J. Bambach, H. Rokita, and D. Kozbor
Targeting a Mimotope Vaccine to Activating Fc{gamma} Receptors Empowers Dendritic Cells to Prime Specific CD8+ T Cell Responses in Tumor-Bearing Mice
J. Immunol.,
November 15, 2009;
183(10):
6808 - 6818.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Huebener, S. Fest, K. Hilt, A. Schramm, A. Eggert, T. Durmus, A. Woehler, A. Stermann, M. Bleeke, B. Baykan, et al.
Xenogeneic immunization with human tyrosine hydroxylase DNA vaccines suppresses growth of established neuroblastoma
Mol. Cancer Ther.,
August 1, 2009;
8(8):
2392 - 2401.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Salcedo, J. A. Hixon, J. K. Stauffer, R. Jalah, A. D. Brooks, T. Khan, R.-M. Dai, L. Scheetz, E. Lincoln, T. C. Back, et al.
Immunologic and Therapeutic Synergy of IL-27 and IL-2: Enhancement of T Cell Sensitization, Tumor-Specific CTL Reactivity and Complete Regression of Disseminated Neuroblastoma Metastases in the Liver and Bone Marrow
J. Immunol.,
April 1, 2009;
182(7):
4328 - 4338.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Wierzbicki, M. Gil, M. Ciesielski, R. A. Fenstermaker, Y. Kaneko, H. Rokita, J. T. Lau, and D. Kozbor
Immunization with a Mimotope of GD2 Ganglioside Induces CD8+ T Cells That Recognize Cell Adhesion Molecules on Tumor Cells
J. Immunol.,
November 1, 2008;
181(9):
6644 - 6653.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Wagner, P. Schulz, A. Scholz, B. Wiedenmann, and A. Menrad
The Targeted Immunocytokine L19-IL2 Efficiently Inhibits the Growth of Orthotopic Pancreatic Cancer
Clin. Cancer Res.,
August 1, 2008;
14(15):
4951 - 4960.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Huebener, S. Fest, A. Strandsby, E. Michalsky, R. Preissner, Y. Zeng, G. Gaedicke, and H. N. Lode
A rationally designed tyrosine hydroxylase DNA vaccine induces specific antineuroblastoma immunity
Mol. Cancer Ther.,
July 1, 2008;
7(7):
2241 - 2251.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Zeng, N. Huebener, S. Fest, S. Weixler, U. Schroeder, G. Gaedicke, R. Xiang, A. Schramm, A. Eggert, R. A. Reisfeld, et al.
Fractalkine (CX3CL1)- and Interleukin-2-Enriched Neuroblastoma Microenvironment Induces Eradication of Metastases Mediated by T Cells and Natural Killer Cells
Cancer Res.,
March 1, 2007;
67(5):
2331 - 2338.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. D. Lum, I. N. Buhtoiarov, B. E. Schmidt, G. Berke, D. M. Paulnock, P. M. Sondel, and A. L. Rakhmilevich
In vivo CD40 ligation can induce T cell-independent antitumor effects that involve macrophages
J. Leukoc. Biol.,
June 1, 2006;
79(6):
1181 - 1192.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Helguera, J. A. Rodriguez, and M. L. Penichet
Cytokines fused to antibodies and their combinations as therapeutic agents against different peritoneal HER2/neu expressing tumors.
Mol. Cancer Ther.,
April 1, 2006;
5(4):
1029 - 1040.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. L. Osenga, J. A. Hank, M. R. Albertini, J. Gan, A. G. Sternberg, J. Eickhoff, R. C. Seeger, K. K. Matthay, C. P. Reynolds, C. Twist, et al.
A Phase I Clinical Trial of the hu14.18-IL2 (EMD 273063) as a Treatment for Children with Refractory or Recurrent Neuroblastoma and Melanoma: a Study of the Children's Oncology Group.
Clin. Cancer Res.,
March 15, 2006;
12(6):
1750 - 1759.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Croce, R. Meazza, A. M. Orengo, L. Radic', B. De Giovanni, C. Gambini, B. Carlini, V. Pistoia, L. Mortara, R. S. Accolla, et al.
Sequential Immunogene Therapy with Interleukin-12- and Interleukin-15-Engineered Neuroblastoma Cells Cures Metastatic Disease in Syngeneic Mice
Clin. Cancer Res.,
January 15, 2005;
11(2):
735 - 742.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. M. King, M. R. Albertini, H. Schalch, J. A. Hank, J. Gan, J. Surfus, D. Mahvi, J. H. Schiller, T. Warner, K. Kim, et al.
Phase I Clinical Trial of the Immunocytokine EMD 273063 in Melanoma Patients
J. Clin. Oncol.,
November 15, 2004;
22(22):
4463 - 4473.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Yotnda, B. Savoldo, N. Charlet-Berguerand, C. Rooney, and M. Brenner
Targeted delivery of adenoviral vectors by cytotoxic T cells
Blood,
October 15, 2004;
104(8):
2272 - 2280.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. C. Neal, J. C. Yang, A. L. Rakhmilevich, I. N. Buhtoiarov, H. E. Lum, M. Imboden, J. A. Hank, H. N. Lode, R. A. Reisfeld, S. D. Gillies, et al.
Enhanced Activity of Hu14.18-IL2 Immunocytokine against Murine NXS2 Neuroblastoma when Combined with Interleukin 2 Therapy
Clin. Cancer Res.,
July 15, 2004;
10(14):
4839 - 4847.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Luo, H. Zhou, M. Mizutani, N. Mizutani, R. A. Reisfeld, and R. Xiang
Transcription factor Fos-related antigen 1 is an effective target for a breast cancer vaccine
PNAS,
July 22, 2003;
100(15):
8850 - 8855.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Halin, V. Gafner, M. E. Villani, L. Borsi, A. Berndt, H. Kosmehl, L. Zardi, and D. Neri
Synergistic Therapeutic Effects of a Tumor Targeting Antibody Fragment, Fused to Interleukin 12 and to Tumor Necrosis Factor {alpha}
Cancer Res.,
June 15, 2003;
63(12):
3202 - 3210.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U. Pertl, H. Wodrich, J. M. Ruehlmann, S. D. Gillies, H. N. Lode, and R. A. Reisfeld
Immunotherapy with a posttranscriptionally modified DNA vaccine induces complete protection against metastatic neuroblastoma
Blood,
January 15, 2003;
101(2):
649 - 654.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Carnemolla, L. Borsi, E. Balza, P. Castellani, R. Meazza, A. Berndt, S. Ferrini, H. Kosmehl, D. Neri, and L. Zardi
Enhancement of the antitumor properties of interleukin-2 by its targeted delivery to the tumor blood vessel extracellular matrix
Blood,
March 1, 2002;
99(5):
1659 - 1665.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Shabat, H. N. Lode, U. Pertl, R. A. Reisfeld, C. Rader, R. A. Lerner, and C. F. Barbas III
In vivo activity in a catalytic antibody-prodrug system: Antibody catalyzed etoposide prodrug activation for selective chemotherapy
PNAS,
June 7, 2001;
(2001)
131187998.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Imboden, K. R. Murphy, A. L. Rakhmilevich, Z. C. Neal, R. Xiang, R. A. Reisfeld, S. D. Gillies, and P. M. Sondel
The Level of MHC Class I Expression on Murine Adenocarcinoma Can Change the Antitumor Effector Mechanism of Immunocytokine Therapy
Cancer Res.,
February 1, 2001;
61(4):
1500 - 1507.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. G. Turner, A. L. Rakhmilevich, L. Burdelya, Z. Neal, M. Imboden, P. M. Sondel, and H. Yu
Anti-CD40 Antibody Induces Antitumor and Antimetastatic Effects: The Role of NK Cells
J. Immunol.,
January 1, 2001;
166(1):
89 - 94.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. F. Ozkaynak, P. M. Sondel, M. D. Krailo, J. Gan, B. Javorsky, R. A. Reisfeld, K. K. Matthay, G. H. Reaman, and R. C. Seeger
Phase I Study of Chimeric Human/Murine Anti-Ganglioside GD2 Monoclonal Antibody (ch14.18) With Granulocyte-Macrophage Colony-Stimulating Factor in Children With Neuroblastoma Immediately After Hematopoietic Stem-Cell Transplantation: A Children's Cancer Group Study
J. Clin. Oncol.,
December 15, 2000;
18(24):
4077 - 4085.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Balicki, R. A. Reisfeld, U. Pertl, E. Beutler, and H. N. Lode
Histone H2A-mediated transient cytokine gene delivery induces efficient antitumor responses in murine neuroblastoma
PNAS,
September 29, 2000;
(2000)
210382997.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
H. N. Lode, R. Xiang, S. R. Duncan, A. N. Theofilopoulos, S. D. Gillies, and R. A. Reisfeld
Tumor-targeted IL-2 amplifies T cell-mediated immune response induced by gene therapy with single-chain IL-12
PNAS,
July 20, 1999;
96(15):
8591 - 8596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. S. Peng, M. L. Penichet, and S. L. Morrison
A Single-Chain IL-12 IgG3 Antibody Fusion Protein Retains Antibody Specificity and IL-12 Bioactivity and Demonstrates Antitumor Activity
J. Immunol.,
July 1, 1999;
163(1):
250 - 258.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. D. Gillies, Y. Lan, K.-M. Lo, M. Super, and J. Wesolowski
Improving the Efficacy of Antibody-Interleukin 2 Fusion Proteins by Reducing Their Interaction with Fc Receptors
Cancer Res.,
May 1, 1999;
59(9):
2159 - 2166.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. N. Lode, T. Moehler, R. Xiang, A. Jonczyk, S. D. Gillies, D. A. Cheresh, and R. A. Reisfeld
Synergy between an antiangiogenic integrin alpha v antagonist and an antibody-cytokine fusion protein eradicates spontaneous tumor metastases
PNAS,
February 16, 1999;
96(4):
1591 - 1596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Shabat, H. N. Lode, U. Pertl, R. A. Reisfeld, C. Rader, R. A. Lerner, and C. F. Barbas III
In vivo activity in a catalytic antibody-prodrug system: Antibody catalyzed etoposide prodrug activation for selective chemotherapy
PNAS,
June 19, 2001;
98(13):
7528 - 7533.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Balicki, R. A. Reisfeld, U. Pertl, E. Beutler, and H. N. Lode
Histone H2A-mediated transient cytokine gene delivery induces efficient antitumor responses in murine neuroblastoma
PNAS,
October 10, 2000;
97(21):
11500 - 11504.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract
