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Introduction
The administration of high-dose recombinant interleukin
2 (IL-2) to patients has been reported to mediate the regression of
tumors in selected patients with metastatic melanoma, kidney cancer, and non-Hodgkin's
lymphoma[1_5]. However, the potent antitumor activity of IL-2 is
often achieved at the expense of unacceptable
toxicity[6,7]. Minimizing IL-2 toxicity requires both
the prevention of the systemic side-effects of IL-2 and the locally restricted enrichment of IL-2 activity to the tumor
sites[8,9]. Direct injection into
tumors[10_13] and cytokine gene
therapy[7,8,14_16] were considered as approaches to solve this problem.
However, direct injection requires the localization of the tumor but it seems impossible to directly inject IL-2 into micrometastases.
Gene therapy approach requires that tumor cells be removed from the patients, transduced and reintroduced into patients,
which is sophisticated to manipulate. Tumor-specific antibodies genetically fused to IL-2 provide an alternative approach to
improve the therapeutic index of
IL-2[17_19]. The underlying principle of antibody-IL-2 molecules is based on the targeting of
potent cytokines to the tumor microenvironment, where they elicit an immune response and thus destroy
tumors[1,6,20]. In the past decade, several groups have reported
different antibody_IL-2
fusions[17,21,22]. These novel proteins were shown to retain
both antibody and cytokine functions and to exhibit
superior anticancer activities as compared with equivalent amounts
of free IL-2 and antibodies.
A humanized anti-ErbB2 antibody, Herceptin has
succeeded in clinical trials for the treatment of ErbB2-expressing
metastatic breast cancer[23_25]. The fusion of IL-2 to an
ErbB2-specific antibody may enhance its efficacy. Fusion proteins
of intact antibodies and IL-2 have been well investigated
and have been shown to mediate the eradication of
established metastatic tumors in several animal
models[17_19,22]. In previous studies, we constructed a different type of fusion
protein (HFI) consisting of an anti-ErbB2 single chain
antibody (scFv), Fc of human IgG1 and
IL-2[26]. IL-2 was fused to the C terminus of anti-ErbB2 scFv_Fc. The molecular
weight of the fusion protein is about two-third of the whole
antibody_IL-2 fusion protein, which may confer the fusion
protein better penetration property and higher
expression level[27]. In the present study,
our in vitro and in vivo data demonstrate that the fusion protein exhibits high efficiency,
both in mediating antibody-dependent cell-mediated
cytotoxicity (ADCC) activity in vitro and significant antitumor
activity in tumor xenograft models.
Materials and methods
Tumor cell lines and animals The tumor cell lines
used were human ovarian carcinoma cell line SKOV3 and
human breast carcinoma cell lines SKBR3 and MCF7. The
cells were maintained in high-glucose Dulbecco's
modified Eagle's medium (DMEM) or RPMI-1640 supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan,
UT, USA ) until ready for use.
Female Balb/C athymic nude mice were purchased from
the Institute of Laboratory Animal Science, Chinese
Academy of Medical Science (Beijing, China) and housed in
specific, pathogen-free conditions in the Good Laboratory
Practice facility. For all the studies, the mice were allowed to
acclimate at least 3 d.
Construction of anti-ErbB2 scFv_Fc_IL-2 fusion
protein Anti-ErbB2 scFv, human IgG1 Fc region, and
human IL-2 genes were cloned into plasmid pCI-dhfr (Promega,
San Luis Obispo, CA, USA) as previously
described[26]. The schematic construct of HFI is shown in Figure 1. The
plasmid containing the anti-ErbB2 scFv_Fc fusion gene (named
scFv_Fc) was also constructed. Chinese hamster ovary
(CHO) cells stably expressing HFI were generated by
transfection of the plasmids and selection for dhfr+transformats
in DMEM supplemented with 10% dialyzed FBS (Gibco,
Carlsbad, CA, USA) and methotrexate. The fusion proteins
from the cell culture medium were purified by affinity
chromatography as described
previously[26]. The size of the fusion proteins was analyzed in reducing conditions on
SDS_PAGE and in native conditions, in which the SDS and
reducing agent (DL-Dithiothreitol, DTT) were omitted from the
standard Laemmli SDS protocol and in the sample buffer, by
laser desorption time-of-flight mass spectrometry
(MALDI_TOF_MS; Bruker_Franzen, Bremen, Germany).
Cytotoxicity assays The efficacy of the fusion
protein in mediating tumor cell lysis was determined by
colorimetric lactate dehydrogenase (LDH) release assays.
4×103 SKBR3, SKOV3, and MCF7 cells in 100 µL LDH as
the target cells were added to 96-well flat-bottom plates,
respectively, and incubated overnight. Subsequently,
peripheral blood mononuclear cells (PBMC) isolated from
the blood of healthy donors by Ficoll density gradient
centrifugation as the effector cells were added to each well at
effector-to-target (E:T) ratios of 5:1, 10:1, 20:1, and 40:1
in a final volume of 100 µL/well with the anti-ErbB2
scFv_Fc_IL-2 fusion protein (HFI) at the final concentration of
15 ng/mL. Spontaneous release (SR) was set by adding only
target or effector cells; maximum release (MR) was set by
adding 1% Triton X-100 to the target cells. After 4_8 h of
incubation at 37 ºC in 5% CO2, the assays were centrifuged.
The supernatant was transferred to 96-well flat-bottom
plates and incubated with LDH reaction mixture (LDH
Detection Kit, Roche, Mannheim, Germany) for 30 min at
25 ºC. The samples were measured at 492 nm with a
reference wavelength of 620 nm. The percentage of
cytotoxicity was calculated as ([Sample
release_SReffector_SRtarget]/
[MRtarget_SRtarget])×100. RPMI-1640 medium was used as
the control.
For light microscopy, SKBR3 cells were incubated with
PBMC in the medium containing HFI at a final concentration
of 15 ng/mL in 96-well plates at 37 °C for 12 h. The cells were
then directly viewed under a light microscope (Nikon,
Yokohama, Kanagawa, Japan).
Lymphokine-activated killer cell
generation Human PBMC at a cell concentration of
2.5×106_5×106/mL in 50
mL flasks was stimulated with recombinant interleukin 2
(rIL-2) at 500 IU/mL in RPMI-1640 medium supplemented
with 2 mmol/L glutamine, 100 U/mL penicillin, 100
mg/mL streptomycin, and 20% FBS. The cells were
cultured for 5_7 d in a humidified incubator with 5%
CO2 at 37 °C. The cells were harvested and viability was
determined by the trypan blue dye exclusion method.
In vivo antitumor activity Six to seven-week-old
female Balb/C athymic nude mice were sc injected into the
right flank with 1×107 SKOV3 cells. The mice were treated
intravenously with recombinant IL-2 (10 000 IU), HFI (5,
2.5, and 1.25 µg, respectively), scFv_Fc (5 µg) in combination
with 1×106 lymphokine-activated
killer (LAK) cells, or phosphate-buffered saline (PBS) twice weekly. In each group, 7
or 8 mice were used. The mice were monitored daily and
tumor volume was measured with a caliper twice a week,
using the formula
volume=length×width2/2. At the end of
the experiments (after treatment for 21_35 d), the mice were
killed. The organs (spleen, heart, lung, liver, and kidney)
and primary tumors were removed, fixed in formalin, and
examined histologically. Flow cytometry
(FACS) was used to examine the effector cell profile, the blood was collected from
the mice by retro-orbital bleeding in heparinized Eppendorf
microcentrifuge tubes. FITC conjugated anti-human CD3
and anti-human CD16 antibodies were added at a final
concentration of 2 µg/mL in 100 µL volume and incubated on ice
for 20 min. The stained cells were washed 3 times with
ice-cold PBA (PBS, 2% BSA, 0.1% NaN3) and analyzed on a
FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA),
using CELLQUEST software (BD Biosciences, San Jose, CA,
USA).
Histology and immunohistochemistry Tissue
samples were fixed in 4% buffered paraformaldehyde and
embedded in paraffin. Tumor tissues were analyzed by
hematoxylin_eosin (HE) staining and immunohistochemistry.
Briefly, for the immunohistochemical analysis, the
paraffin-embedded sections were dewaxed and rehydrated.
Endogenous peroxidase activity was quenched by 3%
hydrogen peroxide in distilled water for 5_10 min and then washed
in PBS before immunohistochemical staining. The Streptavidin_Biotin method was used to detect ErbB2
expression in the tumor tissues. Primary antibodies (mouse
monoclonal antibodies against ErbB2), the biotinylated
antimouse antibody, and the streptavidin peroxidase reagent
were purchased from Beijing Zhongshan Golden Bridge
Biotechnology (Beijing, China). Antigen retrieval was
achieved by microwave treatment. Sections were
sequentially incubated with the primary antibody, the biotinylated
antimouse antibody, and the streptavidin peroxidase reagent.
Peroxidase activity was detected with 3,3'-diaminobenzidine
(DAB) solution, and the sections were weakly counterstained
with hematoxylin, washed, dehydrated with alcohol and
xylene, and mounted with coverslips. Omission of the
primary antibody was used as negative controls.
Toxicity studies Six-week-old female Balb/C athymic nude
mice (n=4) were given 1 single iv injection of 0, 5, 10, or 20 µg
HFI diluted in a volume of 100 µL PBS. The mice were
monitored daily and toxicity was measured in terms of weight loss
and mortality.
Statistical analysis Data were expressed as mean±SD.
For the cytotoxicity assay, the data were compared
by t-test using standard statistical software, SPSS version 11.0
(Chicago, IL, USA). For comparisons among the groups in
the experiments in vivo, an ANOVA test was performed.
P<0.05 was considered statistically significant.
Results
Characterization of fusion proteins The
antigen-binding and IL-2 activities of the fusion proteins were determined
by FACS and ELISA as described previously (data not
shown)[26]. HFI specifically and efficiently bound to SKBR3 cells
expressing high levels of ErbB2, but bound less efficiently
to MCF7 cells expressing low levels of
ErbB2[28], indicating that the
fusion of anti-ErbB2 scFv_Fc to the amino terminus
of IL-2 did not interfere with the ability of the antibody to
recognize the ErbB2 antigen. To determine the molecular
mass and assembly of the fusion proteins, the purified
proteins were analyzed by SDS_PAGE and MALDI_TOF_MS.
In the presence of reducing agents, the fusion proteins
migrated with an apparent molecular mass of ~70 kDa, the
expected molecular mass of the fusion protein, as previously
determined (data not shown)[26]. The MALDI_TOF_MS
indicated the purified HFI (Figure 2). The molecular weight of
HFI in a monovalent form was found to be 70 871
m/z and that in a bivalent form was 141393 m/z. The results indicated
that the fusion of IL-2 to anti-ErbB2 scFv_Fc did not
appear to alter the assembly and secretion of the bivalent form of
the scFv_Fc_IL-2 fusion protein.
Cytotoxicity studies HFI was evaluated for its ability to
mediate ADCC by LDH assays against ErbB2-expressing
cells. Examples of killing assays are shown in Figure 3A_3C.
At a concentration of 15 ng/mL and all E:T ratios, the fusion
protein mediated the efficient lysis to SKBR3 and SKOV3
cells, which expressed high levels of
ErbB2[29,30] (P<0.01). Both
cells were killed to a greater extent by HFI-mediated lysis
than MCF7 cells expressing low levels of ErbB2,
demonstrating that HFI was effective against the ErbB2-overexpressing
cell lines in vitro.
Morphological observations clearly revealed the
binding and lysis of effector cells to tumor cells mediated by HFI
(Figure 3D and 3E). An extremely swollen SKBR3 cell was
tightly surrounded by the effector cells after 12 h incubation
(Figure 3E). The dying or dead tumor cells were easily
observed in the incubation system. In contrast, the effector
cells largely appeared healthy with intact cell membranes.
In vivo antitumor activity After demonstrating that HFI
had in vitro biological activity, the in
vivo antitumor activity was investigated
using a SKOV3 animal model. A total of
1×107 SKOV3 cells in 0.15 mL PBS were
injected sc into the right flank of the mice on d 1. A direct comparison between
free IL-2 and HFI was performed on d 3 after sc tumor cell
implantation. The therapy groups consisted of mice
(8 animals/group) receiving iv injections of PBS, IL-2 (10 000 IU),
and HFI (5 µg) in combination with LAK cells twice weekly.
1 µg HFI contains ~2000 IU of IL-2 activity as determined by
in vitro proliferative assays with IL-2-dependent
T-cells[26]. The data demonstrated a dramatic improvement in efficacy
with complete regression in all mice treated with 5 µg HFI
twice weekly for 21_25 d (P<0.01) (Figure 4A). The improved
efficacy is likely attributable to the increased half-life
and improved targeting of IL-2.
To determine the antitumor activity of HFI at the
decreased doses and prolonged treatment, therapy was started
in groups of mice treated with PBS or with 5, 2.5, or 1.25 µg
HFI, respectively. Injections were repeated twice weekly for
35 d. After 29 d treatment, tumors started to regrow
(Figure 4B_4D). Only 1 of the tumors in the group treated with 5 µg
HFI was regressed. One mouse in the group treated with
1.25 µg HFI died. The therapeutic effect observed in the
mice treated with the 5 µg dose was considerably better
than that observed with the 2.5 (P<0.01) and 1.25 µg doses
(P<0.01).
To compare the therapeutic efficacy of HFI with that of
scFv_Fc and to investigate the effect of HFI on larger tumors,
injections of PBS, scFv_Fc (5 µg), or HFI (5 µg) were started
when the tumors reached ~285 mm3 in size on d 6 after tumor
cell implantation. Treatment with HFI arrested the growth of
tumors approximately 6 d after the beginning of the
treatment compared with the controls (P<0.05), whereas scFv_Fc
exhibited no significant effect compared with PBS and free
IL-2 (P>0.05); the tumors developed quickly after treatment
with scFv_Fc for 15 d. (Figure 4E and 4F).
Effector cell profile The effector cell population was
derived from normal human PBMC after culturing for 7 d in
medium containing IL-2. Both T cell and natural killer cell
(NK) populations are expected to be activated
by this procedure. Human CD3+ and
CD16+ cells were detected from the peripheral blood of mice in all doses of the HFI treated
groups (Table 1).
Toxicity Treatment with HFI was well tolerated at the 5
µg dose and no significant weight loss was apparent. A
transient weight loss (<5%) was observed, but the toxicity
appeared to be moderate. The greater weight loss (~6%)
occurred at the 20 µg dose, which was 4-fold
greater than the one used in the therapy experiments. All the mice survived
the experiment.
Histology and immunohistochemistry Histologically, the
PBS-treated tumors consisted of large pleomorphic cells that
had large dark nuclei and increased basophilic cytoplasm
with some mitoses. Many infiltrations of the tumor cells into
the adjacent muscle tissues were often observed (Figure 5A).
It is noteworthy that the tumors treated with HFI were
surrounded by the thin fibrous capsule and a large amount of
infiltrating lymphocytes (Figure 5B and 5C). This is in
contrast to the tumors, where no lymphocyte infiltrate was
observed from mice when treated with either PBS or free IL-2.
In the mice of all the groups, no pathological findings were
seen in the spleen, kidney, lung, and heart. A histological
examination of livers from the mice treated with HFI revealed
the presence of inflammatory foci and focal hepatocyte
necrosis. Similar characteristics were found in the study of
antitumor activity of targeted IL-12 and have been described
as IL-12-induced hepatotoxicity[32].
The tumors from the mice (6 or 7 for each group) were
analyzed by immunohistochemistry for ErbB2 expression.
The results demonstrated that ErbB2 was strongly
expressed in the control tumors (Figure 6A). However, the
expression of ErbB2 in the tumor tissues was remarkably
decreased or even disappeared after 5 µg HFI treatment
(Figure 6B and 6C).
Discussion
IL-2 is an important pleiotrophic cytokine and exhibits a
wide variety of biological activities, including the
stimulation of antitumor effector cells, such as cytotoxic T
cells[33_35], NK
cells[33,36_39], and
macrophages[7,15,16]. It also influences
dendritic cell interactions with various effector cell
populations, which may elicit further antitumor involvement
with subsequent development of tumor-specific T cell
responses[16]. It has been reported that IL-2 treatment can
augment the activation of pre-existing antigen-specific T cells,
enhance their recognition and destruction of neoplastic
tissue, and activate NK cells[1,37,40]. However, systemic
cytokine therapy frequently causes severe problems with
toxicity that makes it impossible to achieve an effective dose
at the tumor sites. Increasing local cytokine
concentration and limiting generalized
toxicity have been the goals pursued by researchers and clinicians. In recent years, the
antibody has become a specific delivery vehicle to selectively
target IL-2 to metastatic/residual
nodules. The specific targeting has been expected to elicit the local activation of NK cells at
tumor sites to provide more effective tumor
destruction[19_22].
Several antibody_cytokine fusion proteins have been
developed. In the preclinical trials using murine models,
antibody_cytokine fusion proteins have shown to be very
effective antitumor agents. Most studies have focused on
IL-2 as the effector molecule and whole antibodies were
generally used as the targeting vehicles. The production of a
whole antibody_cytokine fusion protein requires the co-expression of a heavy chain usually fused to IL-2 and a light
chain in a single host. It does not guarantee that both heavy
chain and light chain are present in equimolar concentrations.
In the present study, we constructed an anti-ErbB2
scFv_Fc_IL-2 fusion protein, HFI. The recombinant human IL-2
gene is fused with the anti-ErbB2 scFv_Fc gene at the
C-terminal. The fusion protein was expressed as a single
peptide and folded as a homodimer formed by the covalently
linking of Fc portions leading to a bivalent molecule. The
biological activities of both antibody and IL-2 were well
maintained. HFI was demonstrated as potent to initiate a
cytotoxic activity of previously unstimulated PBMC against
human breast and ovarian cancer cells at low
effector-to-target ratios in experiments in
vitro. It was more effective in killing SKBR3 and SKOV3 cells expressing ErbB2 highly than
MCF7 cells expressing ErbB2 at lower
level[29,30], suggesting that the killing effect mediated by HFI was dependent on the
expression level of ErbB2 molecules on the target cells.
To determine the efficacy of the fusion protein
in vivo, the xenograft models of human ovarian carcinoma tumors in
nude mice were treated with engrafted human effector cells
and fusion proteins. The analysis by FACS showed that
both human T and NK cells were present in the peripheral
blood of mice. NK cells may play the major role in
this process since they are more potently activated by high
doses of IL-2 than T cells cultured in the absence of their cognate
antigens. Nonetheless, a role of human T cells cannot be
ruled out. Potent antitumor effects mediated by both NK
and T cells have been reported in a severe combined
immunodeficiency mouse transplantation
model[41]. In this study, significant antitumor activity of the fusion protein was
demonstrated in an in vivo environment. In the mice treated with
HFI, the tumor environment became infiltrated with
lymphocytes. In addition, the inhibition effect on tumor
growth in mice was observed when the treatment with HFI
was started after large tumors had already established,
although complete eradication of tumors
was not achieved. No signs of overt
toxicity were seen in the mice injected with
twice-weekly doses of the fusion proteins. Notably,
splenomegaly was observed in most of the HFI-treated mice. It
has been reported that the doses required for an adjuvant
effect of CD40 mAb when mixed with antigen are highly
reactogenic, leading to splenomegaly and polyclonal
antibody production[31]. The speculative adjuvant effect caused
by HFI may still need to be determined.
ErbB2 overexpression underlies enhancement in
pro-liferative, prosurvival, and metastatic signaling. ErbB2
mediated signaling also confers resistance to anticancer
agents, such as cisplatin, paclitaxel, doxorubicin, etoposide,
and radiation[42_44]. In breast cancer, ErbB2 overexpression
is a significant negative prognostic indicator for a variety of
therapies. Patients with ErbB2-overexpressing tumors have
shown a significantly lower overall survival rate and shorter
relapse time than those with ErbB2-negative
tumors[45,46]. Therapeutic antibody Herceptin has been shown to
dephosphorylate and downregulate ErbB2 levels, block receptor
signaling, and exhibit clinical efficacy against breast
cancer[25,47]. The downregulation of ErbB2 by Herceptin has been shown
to impact cell sensitivity to chemotherapeutic agents,
suppress ErbB2-induced malignant phenotypes, and results in
the recruitment of the cbl protein, ubiquitination, and
inducing the proteasome-dependent degradation of
ErbB2[48]. It is noteworthy that ErbB2 expression in the tumors diminished
after 5 µg dose HFI treatment in this study. However, the
efficacy of ErbB2-targeting inhibitors is correlated with the
ErbB2 overexpression level. The largest effects of these
therapies are seen in the cell lines expressing the highest
levels of ErbB2[28]. It was reported that the effect of Herceptin
was restricted to BT474 and SKBR3 cells, which express ErbB2
highly, but not in MCF-7 cells with low levels of ErbB2. It may
explain that the tumors in the mice resumed growth after
treatment by HFI for approximately 1 month in the present
study. The similar results were also observed by treatment
with Herceptin[49]. Herceptin improved sensitization of
ErbB2-overexpressing breast cancer cells to Taxol and several
chemotherapeutic-induced apoptosis, cytotoxicity, and tumor
growth inhibition[50_53]. Thus, pretreatment of the
therapeutic antibody or antibody_cytokine fusion protein may
enhance the cytotoxicity of chemotherapeutic agents and
benefit the treatment of ErbB2-overexpressing breast carcinomas.
The mechanisms of ErbB2 downregulation by HFI are not
clear.
In conclusion, the genetically engineered anti-ErbB2
scFv_Fc_IL-2 fusion protein retains ErbB2 specificity and
IL-2 biological activity. The fusion proteins mediated
efficient ADCC in vitro and exhibited significant antitumor
activity in the tumor model using SKOV3 ovarian cancer cells.
It is simple to express such a bivalent fusion protein. It has
been found that NK cells were not deficient in patients with
solid and hematological malignancies, even after high-dose
chemotherapy. With the efficient stimulation by targeted
IL-2, NK cells may elicit an antitumor response in patients
who are deficient in T cells following high-dose
chemo-therapy.
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