Folate-immunoglobulin G as an anticancer therapeutic antibody

Article abstract of DOI:10.1021/bc900545h

Folate receptor-α (FR) is a promising cellular marker for tumor-specific drug delivery. Conjugation of folic acid to therapeutic and imaging agents has been shown to enhance their delivery to FR (+) cancer cells in vitro and in tumor-bearing mice via an FR-mediated cellular uptake mechanism. In this study, immunoglobulin G (IgG) was conjugated to folate and evaluated as a therapeutic antibody against folate receptor (FR)-positive tumors. Murine IgG (mIgG) was conjugated to folate via an amide bond to yield folate-conjugated mIgG (f-mIgG) that contained an average of ～2.6 folates per molecule. Selective uptake of f-IgG by FR (+) tumor cells was determined by fluorescence microscopy and by flow cytometry. Lysis of L1210JF cells by NK cells from murine donors was increased 1.4-9.0-fold at the effector:target (E:T) ratio of 25:1, relative to control mIgG. In mice bearing L1210JF tumors, f-mIgG was found to significantly inhibit tumor growth and to have prolonged the median survival time (MeST). Significantly, the antitumor efficacy of f-mIgG was greatly increased when combined with liposomal G3139, an 18-mer phosphorothioate oligonucleotide. In fact, the combination resulted in a 100% cure rate among the tumor-bearing mice. Injection of f-mIgG significantly increased serum INF-- and IL-6 level in mice compared with mIgG and dramatically increased serum INF-- and IL-6 level when combined with liposomal G3139. These results suggested that f-IgG, a novel immunotherapy agent, has potent activity as a therapeutic antibody to the FR-positive cancer, and the therapeutic activity is enhanced by immunomodulatory agents.

Full text of DOI:10.1021/bc900545h

Bioconjugate Chem. 2010, 21, 961–968
961
Folate-Immunoglobulin G as an Anticancer Therapeutic Antibody
Hong Li,†,‡ Yanhui Lu,†,‡ Longzhu Piao,‡ Jun Wu,‡ Xiaojuan Yang,‡,§ Sri Vidya Kondadasula,|,⊥
William E. Carson,|,⊥,# and Robert J. Lee*,‡,§,⊥
Division of Pharmaceutics, NSF Nanoscale Science and Engineering Center (NSEC), Department of Molecular Virology,
Immunology, and Medical Genetics, OSU Comprehensive Cancer Center (OSUCCC), and Department of Surgery, The Ohio
State University, Columbus, Ohio 43210. Received December 9, 2009; Revised Manuscript Received February 5, 2010
Folate receptor-R (FR) is a promising cellular marker for tumor-specific drug delivery. Conjugation of folic acid
to therapeutic and imaging agents has been shown to enhance their delivery to FR (+) cancer cells in vitro and
in tumor-bearing mice via an FR-mediated cellular uptake mechanism. In this study, immunoglobulin G (IgG)
was conjugated to folate and evaluated as a therapeutic antibody against folate receptor (FR)-positive tumors.
Murine IgG (mIgG) was conjugated to folate via an amide bond to yield folate-conjugated mIgG (f-mIgG) that
contained an average of ∼2.6 folates per molecule. Selective uptake of f-IgG by FR (+) tumor cells was determined
by fluorescence microscopy and by flow cytometry. Lysis of L1210JF cells by NK cells from murine donors was
increased 1.4-9.0-fold at the effector:target (E:T) ratio of 25:1, relative to control mIgG. In mice bearing L1210JF
tumors, f-mIgG was found to significantly inhibit tumor growth and to have prolonged the median survival time
(MeST). Significantly, the antitumor efficacy of f-mIgG was greatly increased when combined with liposomal
G3139, an 18-mer phosphorothioate oligonucleotide. In fact, the combination resulted in a 100% cure rate among
the tumor-bearing mice. Injection of f-mIgG significantly increased serum INF-γ and IL-6 level in mice compared
with mIgG and dramatically increased serum INF-γ and IL-6 level when combined with liposomal G3139. These
results suggested that f-IgG, a novel immunotherapy agent, has potent activity as a therapeutic antibody to the
FR-positive cancer, and the therapeutic activity is enhanced by immunomodulatory agents.
G3139 (oblimersen or Genasense) is an 18-mer phospho-
rothioate oligonucleotide that was designed as an antisense agent
against antiapoptotic factor bcl-2 (19-21). G3139 contains 2
CpG dinucleotides (22-24) and has been shown to induce a
potent cytokine response, presumably through activation of toll-
like receptor 9 (TLR9) (25, 26). Liposomal encapsulation of
G3139 has recently been shown by us to further enhance the
immunomodulatory effect of G3139 (27).
The objective of this study was to evaluate the therapeutic
efficacy of folate conjugated IgG (f-IgG) as a therapeutic
antibody against FR(+) tumors. F-mIgG conjugates was syn-
thesized and evaluated in vitro as well as in vivo. Furthermore,
the combined effect of f-mIgG and liposomal G3139 was also
examined.
INTRODUCTION
Folate receptor-R (FR) is a promising cellular marker for
tumor-specific drug delivery, because it is up-regulated in many
types of human epithelial cancers. These include tumors arising
in the ovary, lung, kidney, mesothelium, head and neck, and
brain (1-5). Conjugation of folic acid to therapeutic and
imaging agents has been shown to enhance their delivery to
FR(+) cancer cells in vitro and in tumor-bearing mice via an FR-
mediated cellular uptake mechanism (6-8) and are currently being
developed in the clinic by Endocyte Inc. Some folate conjugates
have shown significant antitumor activities as immunotherapeutic
agents (9-12). For example, folate-fluorescein was shown to
eliminate FR(+) tumors in fluorescein-immunized mice (11, 12).
Moreover, co-administration of interleukin 2 (IL-2) and inter-
feron-R (IFN-R) was shown to enhance the therapeutic efficiency
of the folate-fluorescein conjugate (11). In other studies, anti-
FR-R monoclonal antibodies such as Mov-18 (13, 14) and
MORAb-003 (Farletuzumab) (15) have been reported to target
ovarian cancer and have undergone clinical evaluation as
therapeutic agents with promising initial results. In addition, a
folate-conjugated anti-TCR antibody was shown to re-target
cytotoxic T lymphocytes to FR(+) tumors resulting in significant
therapeutic activity (16-18).
EXPERIMENTAL SECTION
Materials. Murine IgG (mIgG) was purchased from Equitech-
Bio Inc. (Kerrville, TX). Folic acid, fluorescein isothiocyanate
(FITC isomer I), N,N′-dicyclohexylcarbodiimide (DCC), and
N-hydroxysuccinimide (NHS) were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). G3139 was synthesized
by Alpha DNA (Montreal, CA). Phosphatidylcholine from egg
yolk (EPC), 3ꢀ-[N-(N′,N′-dimethylaminoethane)carbamoyl] cho-
lesterol hydrochloride (DC-Chol), and methoxy-poly(ethylene
glycol) (MW ) 2000) distearoyl phosphatidylethanolamine
(mPEG-DSPE) were purchased from Avanti Polar Lipids
(Alabaster, AL). All chemicals used were of analytical or HPLC
grade. Recombinant IL-2 with specific activity of 1 × 106 IU/
mL was obtained from Hoffmann-La Roche Pharmaceuticals
(Nutley, NJ).
* Address correspondence to: Dr. Robert J. Lee, College of
Pharmacy, 500 West 12th Ave., Columbus, Ohio 43210. Phone: (614)
292-4172; Fax: (614) 292-7766; E-mail: lee.1339@osu.edu.
† These authors contributed equally to this work.
‡ Division of Pharmaceutics.
§ NSEC.
| Department of Molecular Virology, Immunology, and Medical
Genetics.
Cell Lines. KB and K562 cells were purchased from
American Type Culture Collection (Rockville, MD). L1210JF
cells were provided by Dr. Manohar Ratnam at University of
Toledo (Toledo, OH). KB, L1210JF cells were grown in folate-
⊥ OSUCCC.
# Department of Surgery.
10.1021/bc900545h  2010 American Chemical Society
Published on Web 04/29/2010

962 Bioconjugate Chem., Vol. 21, No. 5, 2010
Li et al.
Figure 1. Synthesis of f-IgG conjugate. Conjugation to γ-carboxylic acid in folate is shown in this figure. However, the R-carboxylic acid group
can also be conjugated.
free RPMI 1640 medium, and K562 cells were grown in
RPMI1640 medium (Invitrogen Life Technologies Inc.). The
media were supplemented with 10% heat-inactivated fetal
bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin,
and 100 mg/mL streptomycin. The cells were maintained in a
humidified incubator at 37 °C in 5% CO2.
°C. Target cell lysis was determined by counting 100 µL of
supernatant in a scintillation counter after removal of intact cells
by centrifugation. Spontaneous and maximum lysis were
determined by incubating radiolabeled target cells in the absence
of effector cells or by adding 10% SDS to the radiolabeled target
cells, respectively. The percentage of cell lysis was calculated
by the following formula: Lysis (%) ) 100 × (experimental
release - spontaneous release)/(maximal release - spontaneous
release).
Synthesis of Folate-Conjugated Antibodies. The f-IgG was
synthesized as previously described (28) with minor modifica-
tions. Briefly, folate was reacted with N,N′-dicyclohexylcarbo-
diimide (DCC) and N-hydroxysuccinimide (NHS) in DMSO at
molar ratios of folate/DCC/NHS ) 1/1.5/1.1 for 3 h at room
temperature. The product f-NHS was centrifuged to remove
byproducts and then was added to IgG in a pH 8.0 PBS buffer
solution at a folate-to-IgG molar ratio of 12:1 for 2 h at room
temperature. For the preparation of FITC labeled f-IgG (f-IgG-
FITC) or IgG (IgG-FITC), 5× FITC dissolved in a small volume
of DMSO was added to the protein solution at pH 8. To purify
the conjugates, the reaction mixture was passed through a PD-
10 column equilibrated in PBS (pH 8.0) and the product was
collected in the void volume fractions. Folate content of the
product was determined by UV spectrometry at 371 nm, and
the antibody protein concentration was determined using the
Pierce BCA Protein Assay Kit. A synthetic scheme is described
in Figure 1.
Uptake of f-IgG-FITC by Tumor Cells. The cellular uptake
of f-IgG was determined in FR(+) KB, L1210JF, and FR(-)
K562 cells. Approximately 1 × 106 cells were incubated with
200 nM FITC labeled IgG or f-IgG in 1 mL folate-free
RPMI1640 at 37 °C for 30 min. The cells were then washed
three times with cold PBS containing 1% BSA and examined
by fluorescence microscopy or by flow cytometry. For receptor
blocking studies, 200 µM of free folate was added to media
during f-IgG-FITC exposure.
Antibody Dependent Cellular Cytotoxicity (ADCC)
Mediated by f-IgG in L1210JF Cells. Murine NK cells were
isolated from splenocytes of DBA/2 mice (Charles River,
Wilmington, MA) via mechanical digestion followed by lysis
of red blood cells. Spleenocytes were then incubated with
phycoerythrin-labeled anti-DX5 antibody (BD PharMingen, San
Diego, CA) for 30 min at 4 °C. Following this, the cells were
washed and incubated with antiphycoerythrin antibody-coated
magnetic microbeads (Miltenyi Biotech, Auburn, CA) for an
additional 30 min at 4 °C according to the instructions of the
manufacturer. NK cells were isolated via positive selection
through an LS+ magnetic column (Miltenyi Biotech). Isolated
murine NK cells were >90% DX5+ by FACS analysis. Purified
murine NK cells were plated in 96-well V-bottom plates in 10%
HAB containing glutamine free, folate-free RPMI 1640 medium
supplemented with a low dose of IL-2 (1 nM), and incubated
overnight at 37 °C. For ADCC assays, mIgG or f-mIgG treated,
51Cr-labeled L1210JF tumor cells were added to NK cells at
effector:target (E:T) ratios of 25:1 and 12.5:1 and 6.25:1, and
cytotoxic interactions were allowed to proceed for 4 h at 37
Preparation of Liposomal G3139. Liposomes containing
G3139 were prepared by the EtOH dilution/diafiltration method,
as described previously (27). DC-Chol, egg PC, and mPEG-
DSPE were dissolved in ethanol and mixed at a molar ratio of
30:65:5. The lipid mixture (20 mg/mL) was mixed with
protamine solution in citrate buffer (pH 4.0, 20 mM) and then
slowly added to G3139 solution in citrate buffer (pH 4.0, 20
mM) under vortex mixing to achieve a final ethanol concentra-
tion of 40% and a G3139, lipid, and protamine ratio of 1:12.5:
0.3 (w/w/w). The preparation was then dialyzed against citrate
buffer (pH 4.0, 20 mM) for 1 h and then against HEPES-
buffered saline (HBS, pH 7.5) overnight to remove ethanol. Final
product was stored at 4 °C before use. The mean particle size
was determined by dynamic light scattering on a Nicomp model
370 Submicrometer Particle Sizer (Particle Sizing Systems,
Santa Barbara, CA). Zeta potential (ꢁ) of liposomal G3139 was
measured on a Brookhaven 90plus Particle Analyzer (Holtsville,
NY).
Therapeutic Efficacy in Murine Leukemia Model. Thera-
peutic efficacy in murine leukemia model was evaluated in
DBA/2 mice (Charles River, Wilmington, MA). Female mice
(18-22 g) were injected subcutaneously on day 0 with 1 ×
106 L1210JF cells and then randomized to different treatment
groups to eliminate cage effects. Before leukemia cell inocula-
tion, mice were placed on a folate-free diet (AIN-90G, Dyets,
Bethlehem, PA) for 1 week. Normal rodent chow was not used
because it contains approximately 3.19 mg/kg folic acid, which
leads to supra-physiological serum folate concentration. When
a tumor reached a volume of ∼25 mm3, the mice were treated
with the desired immunotherapy regimens 4 times every 4 days
(q4dx4). In the first treatment regimen, mice were given 100
mg/kg mIgG or f-mIgG intraperitoneally. In the second treat-
ment regimen, mice were treated with 1 mg/kg free G3139 or
liposomal G3139. In the third treatment regimen, mice were
given 100 mg/kg f-mIgG combined with free G3139 or
liposomal G3139. Tumor dimensions were determined by
measuring with a caliper, and the tumor volume (in mm3) was
calculated by the formula 0.5 × (length × width × height).
Mice were sacrificed once the tumor size reached greater than
1500 mm3. Tumor inhibition ratio was calculated by (C - T)/C
× 100%, where T and C represent the mean tumor volume of
the treated and control animals, respectively. Animal survival
was evaluated by Kaplan-Meier analysis and increase-in-

Folate-IgG for Anticancer Therapy
Bioconjugate Chem., Vol. 21, No. 5, 2010 963
Figure 2. Uptake of f-IgG-FITC in KB (A), L1210JF (B), and K562 (C) cells. Cells were treated with 200 nM FITC labeled IgG or f-IgG in
folate-free RPMI1640 at 37 °C for 30 min. For the receptor blocking study, 200 µM of free folate was added to media during f-IgG-FITC exposure.
f-IgG-FITC binding in KB cells by fluorescence microscopy. After treatment, KB cells were photographed in both phase-contrast (upper panels)
and fluorescence (lower panels) mode on a Nikon Eclipse 800 fluorescence microscopy. Uptake of f-IgG-FITC in L1210JF and K562 cells by flow
cytometry. (1) Untreated cells. (2) Cells were treated with IgG-FITC. (3) Cells were treated with f-IgG-FITC. (4) Cells were treated with f-IgG-
FITC and free folate.
lifespan (ILS, %) was calculated by ILS ) (mean survival time
of the treated mice/mean survival time of control mice - 1) ×
100%.
folate conjugation may adversely affect the effector function
of the IgG, a lower conjugation level was desirable. Therefore,
f-IgG with the lower conjugation level was selected for
subsequent studies. The presence of folate on IgG was further
confirmed by MALDI-TOF mass spectroscopy (data not shown).
Serum Cytokine Levels in Murine Leukemia Model. The
serum cytokine levels were investigated in L1210JF tumor-
bearing DBA/2 mice (Charles River, Wilmington, MA). Before
leukemia cell inoculation, mice were placed on a folate-deficient
diet (AIN-90G, Dyets, Bethlehem, PA) for at least 1 week. At
5 days after tumor cells inoculation, the mice were injected
intraperitoneally with 100 mg/kg mIgG and f-mIgG. For the
therapeutic combination study, f-mIgG was intraperitoneally
administered with free G3139 or liposomal G3139. Blood
samples were collected at 2, 6, and 24 h after injection. Serum
was obtained by centrifugation of blood samples at 7000 × g
for 10 min and stored -20 °C until the time of assay. IL-6 and
INF-γ was determined by ELISA Ready-Set-Go kits.
Statistical Analysis. Statistical analysis of survival data was
performed using the log-rank test. Comparisons between groups
were made by Student’s t tests using the MiniTAB software
(Minitab Inc., State College, PA). Difference in survival or
tumor growth were considered significant when the p value was
<0.05.
To evaluate the FR targeting efficiency of f-IgG, tumor cell
uptake of f-IgG-FITC was determined by fluorescence micros-
copy or by flow cytometry. In FR(+) KB cells, f-IgG-FITC
was shown to be taken up much more compared to IgG-FITC
(Figure 2A). The uptake of f-IgG-FITC was blocked by 200
µM free folate (Figure 2A). The results from flow cytometry
were consistent with those obtained by fluorescence microscopy.
In FR(+) murine L1210JF cells, incubation of f-IgG-FITC with
cells resulted in greater fluorescence intensity compared with
that of cells treated with IgG-FITC (Figure 2B). No increased
fluorescence was observed when cells were incubated with
f-IgG-FITC in the presence of 200 µM free folate (Figure 2B).
In contrast, in FR(-) K562 cells, the uptake of f-IgG-FITC and
of IgG-FITC were similar, and the uptake was not affected by
200 µM free folate (Figure 2C). These results indicated that
f-IgG-FITC selectively targeted FR (+) tumor cells and was
taken up via the FR.
F-IgG Enhanced NK Mediated ADCC against L1210JF
Cells. The effects of f-IgG on NK cell lytic activity were
examined in an ADCC assay using L1210JF tumor cells as
targets. Murine NK cells were activated overnight in medium
supplemented with IL-2 (1nM). These cells were then used as
effectors against 51Cr labeled target cells that were treated with
either mIgG or f-mIgG. As shown in Figure 3A, f-IgG markedly
enhanced the ADCC activity of NK cells at all E:T ratios
RESULTS
Characterization of f-IgG Conjugates. The concentrations
of IgG and folate were determined by BCA protein assay and
UV spectrometry at 371 nm, respectively. Conjugation reactions
performed at IgG-to-folate-NHS ratios of 12:1 and 100:1 yielded
preparations containing an average molar ratio of 2.6 folates
per IgG and 9.0 folates per IgG, respectively. Because excessive

964 Bioconjugate Chem., Vol. 21, No. 5, 2010
Li et al.
In order to investigate whether the anticancer activity was
due to FR targeting, anticancer activity of f-mIgG was evaluated
in the FR (-) L1210 murine leukemia model. As shown in Figure
4C, f -mIgG slightly decreased tumor volume by day 23 after
the engraftment of L1210 cells compared with mIgG and PBS,
although no significant differences were observed among
f-mIgG, mIgG, and PBS. The median survival times (MeST)
for PBS, mIgG, and f-mIgG were 25, 28, and 28, respectively.
Similar to data on tumor inhibition, survival time was not
significantly prolonged by treatment of f-mIgG compared with
mIgG (p ) 0.535; by log-rank test). Taken together, these results
suggested that anticancer activity of f-mIgG in FR positive
L1210JF tumor-bearing mice was likely due to the FR targeting.
Therapeutic Efficacy of Liposomal G3139 in Murine
Leukemia Model. Previous studies have shown that the cellular
uptake and the immunomodulatory effect of G3139 can be
significantly enhanced when delivered in a liposomal form (29).
In order to compare the antitumor efficacy of free G3139 and
liposomal G3139, mice carrying L1210JF tumors were treated
with 1 mg/kg of G3139 in free form or in liposomal form. As
shown in Figure 5, tumor-bearing mice treated with liposomal
G3139 had a 45.5% reduction in tumor volume compared with
PBS-treated mice (p < 0.05). In contrast, free G3139 did not
decrease tumor growth compared with PBS. The median
survival times (MeST) for PBS, free G3139, or liposomal G3139
were 24, 36, and 36 days, respectively (Figure 5). However,
there was no significant difference between free G3139 and
liposomal G3139 (p ) 0.252; by log-rank test). These data
suggested that liposomal G3139 was more effective than free
G3139 or PBS in mediating tumor regression but not in
prolonging survival time of mice compared with free G3139.
Synergistic Effect of f-IgG Combined with Liposomal
G3139. Loomis et al. (30) has shown that antitumor efficacy
of G3139 significantly increased when combined with rituximab,
a monoclonal antibody directed against CD20 in a SCID mouse
xenograft model. Since f-IgG is being evaluated as a therapeutic
antibody, we examined the antitumor efficacy of free and
liposomal G3139 coadministered with mIgG or f-mIgG in the
murine L1210JF leukemia model. Treatment of IgG combined
with liposomal G3139 significantly increased MeST of the mice
compared with the treatment of IgG combined with free G3139
(data not shown). With regard to tumor growth inhibition,
liposomal G3139 further increased the antitumor efficacy of
f-IgG (Figure 5A, Table 1). Importantly, all of the mice were
rendered tumor-free after combined treatment with liposomal
G3139 and f-IgG (Figure 5B). In comparison, less impressive
20% and 25% cure rates were observed following treatment
with f-mIgG alone and following combined treatment with
f-mIgG and free G3139, respectively (Figure 5B). These data
indicate a strong synergistic effect between f-IgG and liposomal
G3139.
Figure 3. Effect of f-IgG on ADCC activity against FR-positive
L1210JF cells at different effector:target ratio (A) and different donor
from different mice (B). The NK cell lytic activity was assessed in a
standard 4 h chromium release assay using f-mIgG-coated L1210JF
cancer cells. The percentage of lysis was calculated as described in
Experimental Section. Data represent the mean ( SD (n ) 3).
examined compared with IgG (Figure 3A). Similar results was
observed at E:T ratio of 25:1 for each of the three donors (Figure
3B). However, a relatively large variation was observed among
different donors, presumably due to the variability in effector
cell activities from different mice.
Characterization of Liposomal G3139. Liposomes contain-
ing G3139 was prepared by the EtOH dilution/diafiltration
method. The G3139 liposomes showed mean diameter of 92.4
( 43.6 nm, encapsulation efficiencies of >95%, and zeta
potential of 5.02 ( 0.4 mV.
Therapeutic Activity of f-IgG in the Murine Leukemia
Model. f-IgG was evaluated in the FR (+) L1210JF murine
leukemia model. Mice bearing L1210JF tumors of ∼25 mm3
in size were treated with PBS, 100 mg/kg of mIgG, or 100 mg/
kg of f-mIgG by intraperitoneal injection every 4 days for a
total of 4 injections. The mice treated with f-mIgG had 59.6%
reduction in tumor volume by day 24 after the engraftment of
L1210JF cells compared with mIgG treated mice (p < 0.05)
(Figure 4A). However, tumor-bearing mice treated with mIgG
did not show any difference in tumor volume compared to the
mice treated with PBS. The median survival time (MeST) for
PBS, mIgG, or f-mIgG groups was 24, 27, and 39 days,
respectively (Figure 4B, Table 1). Percentage ILS values are
62.5% for f-mIgG and 12.5% for mIgG. Moreover, 1 out of 5
mice was completely cured following treatment with f-mIgG
(Figure 4B). The data indicated that f-IgG was more therapeuti-
cally effective than IgG or PBS in inhibiting tumor growth and
prolonging animal survival (p ) 0.004 01; by log-rank test).
Serum Cytokine Levels in Murine Leukemia Model. In
order to investigate the effect of cytokine production on
anticancer activity, the serum cytokine was determined in
DBA/2 mice after administration of mIgG or f-mIgG and
combined with free G3139 or liposomal G3139. As shown in
Figure 6, levels of INF-γ peaked at 8 h, while levels of IL-6
peaked at 2 h. After 24 h, all cytokine levels were diminished.
Compared with mIgG, f-mIgG showed significant cytokine
(INF-γ and IL-6) production (Figure 6A,B). In combination with
free G3139 and liposomal G3139, f-mIgG induced 2.3-fold and
6.1-fold higher INF-γ production than f-mIgG alone at 6 h
postinjection, respectively. Similar results were observed from
IL-6 induction in L1210JF murine leukemia model. These data
suggested that increased uptake of f-mIgG might elevate the

Folate-IgG for Anticancer Therapy
Bioconjugate Chem., Vol. 21, No. 5, 2010 965
Figure 4. In vivo efficacy of folate receptor mediated immunotherapy in L1210JF (A,B) and L1210 (C,D) bearing DBA/2 mice. Mice (5-6 per
group) were implanted subcutaneously on day 0 with 1 × 106 L1210JF cells. On day 5 or 7, animals were treated with 100 mg/kg of mIgG or
f-mIgG. The treatment was continued every 4 days for four times. (A,C) Inhibition of tumor growth. Tumor dimensions were measured every 2
days using calipers. (B,D) Survival of L1210JF and L1210 tumor-bearing mice after treatment with mIgG or f-mIgG. PBS was injected as a vehicle
control. (*, P < 0.01 compared with PBS group.)
Table 1. Survival of Mice after Treatment with mIgG and f-mIgG
Alone or in Combination with Free or Liposomal G3139 (L-G3139)
in L1210JF Tumor-Bearing Micea
preimmunization of the animal and in vivo coupling of the
folate-hapten and the endogenous antihapten antibody, which
may present an challenging issue. The f-IgG strategy is more
analogous to FRR monoclonal antibodies such as MOv-18
(13, 14) and Morab-003 (15) currently under clinical develop-
ment. Compared with FRR monoclonal antibodies, f-IgG targets
both FR subtypes, and the underlying IgG used is human and
polyclonal, which may be less immunogenic.
f-IgG showed much stronger antitumor effect than IgG in
the FR(+) L1210JF tumor-bearing mice (Figure 4A,B) but not
in the FR(-) L1210 tumor-bearing mice (Figure 4C,D). These
results presumably were due to FR-mediated tumor cell target-
ing. Tissue toxicity and splenomegaly was not founded by
treatment of f-IgG in tumor-bearing mice. Selective folate
receptor binding was also observed in the FR(+) L1210JF and
KB cells in vitro (Figure 3). Besides folate receptor binding,
Fc receptor (FcR) is critical in f-IgG mediated immunotherapy
(31). FcR-expressing immune cells that recognize a constant
(Fc) region of IgG and induce immune response such as ADCC,
phagocytosis, neutrophil activation, and the paradoxical inhibi-
tion of B cell activation (31-34). The potential role of NK cell-
mediated ADCC of f-IgG was examined. The results showed
that lysis of L1210JF tumor cells by NK cells was increased
1.4-9.0-fold by treatment of f-IgG as compared with IgG
(Figure 3). In contrast, f-IgG did not bind to FR(-) cell lines
(e.g., A549 human alveolar basal epithelial) and did not enhance
their lysis by NK cells (data not shown). These results suggested
tumor inhibition MeST ILSc
treatment
ratiob (%)
(days) (%)
Log-rank p value
PBS
mIgG
f-mIgG
G3139
L-G3139
f-mIgG+G3139
f-mIgG+L-G3139
0
8.4
59.6
5.6
45.5
83.1
100.0
24
27
39
36
36
0
-
12.5 0.901
62.5 0.00401
50.0 0.0209
50.0 0.00804
70 191.7 0.000495 (0.198)d
∞
∞
0.000495 (0.00432)d
a n ) 6 for each treatment group. b Tumor inhibition ratio was
calculated by (C - T)/C × 100%, where T and C represent the mean
tumor volume of the treated and control mice, respectively. c ILS(%)
was calculated by (MeST of the treated mice/MeST of control mice -
1) × 100%, where MeST represents the mean survival time. d The p
value was calculated in comparison to f-mIgG.
level of cytokines and combined with liposomal G3139 more
potently increased the level of cytokines.
DISCUSSION
FR is a promising target for tumor-specific drug delivery. In
this study, we describe a novel immunotherapy agent that
combines the FR-targeting properties of folate and the effector
functions of the IgG. Although folate-hapten’s activities have
been reported previously (11, 12), that strategy involves

966 Bioconjugate Chem., Vol. 21, No. 5, 2010
Li et al.
Figure 5. In vivo antitumor efficacy of G3139 in L1210JF bearing
DBA/2 mice. Mice (6 per group) were implanted subcutaneously on
day 0 with 1 × 106 L1210JF cells. On day 5, animals were treated
with with 1 mg/kg of free G3139 or liposomal G3139. The treatment
was continued every 4 days for four times. (A) Inhibition of tumor
growth. Tumor dimensions were measured every 2 days using caliper.
(B) Survival of L1210JF bearing mice after treatment with free G3139
or liposomal G3139. PBS was injected as a vehicle control. (*; P <
0.05 compared with PBS group.)
Figure 6. In vivo antitumor efficacy of f-mIgG combined with free
G3139 or liposomal G3139 in L1210JF bearing DBA/2 mice. Mice (6
per group) were implanted subcutaneously on day 0 with 1 × 106
L1210JF cells. On day 5, animals were treated with 100 mg/kg of
f-mIgG alone or combined with 1 mg/kg of free G3139 or liposomal
G3139. The treatment was continued every 4 days for four times. (A)
Inhibition of tumor growth. Tumor dimensions were measured every
2 days using caliper. (B) Survival of L1210JF bearing mice after
treatment with free G3139 or liposomal G3139. PBS was injected as
a vehicle control. (*; P < 0.01 compared with PBS group.)
that NK-mediated ADCC was FR-specific. Moreover, we
demonstrated enhanced INF-γ production by NK cells when
NK cells were co-cultured with f-IgG coated FR(+) tumor cells
(35). Consistent with this, high levels of INF-γ and IL-6 were
observed in the FR(+) tumor-bearing mice after administration
of f-IgG compared with IgG (Figure 6), indicating that increased
binding of f-IgG to tumor might elevate the level of cytokines.
These results suggested that f-IgG might form a bispecific
molecular bridge between the FR(+) tumor cells and immune
effector cells that mediates tumor cell killing, through NK cell
ADCC and cytokine production by NK cells, i.e., the same
mechanism of a therapeutic antibody. A similar mechanism was
reported by Lu et al. with a combination of a folate-hapten
conjugate and endogenous antihapten antibody (11, 12). There-
fore, the potent anticancer activity of f-IgG in FR(+) tumor-
bearing mice observed was likely the result of FcR mediated
ADCC and cytokine production, stimulated by f-IgG coated
tumor cells via the bispecific molecular bridge. In addition,
activated macrophages are known to express folate receptor
(36, 37). Recently, Turk and Low showed that livers and spleens
of tumor-bearing mice preferentially accumulated folate-targeted
liposomes, presumably due to the FR-mediated targeting of
agents to macrophages in livers and spleens (38, 39). Therefore,
cytokine production by f-IgG accumulated macrophages in liver
and spleen may also contribute to the anticancer activity of f-IgG
in tumor-bearing mice. This mechanism remains to be inves-
tigated in the future studies.
G3139 was originally designed as a human bcl-2 mRNA
antisense and has been investigated in phase I to III clinical
trials as a monotherapy or combination with chemotherapy for
treatment of chronic lymphocytic leukemia (CLL) (40, 41).
Importantly, G3139 contains two CpG motifs, which have been
shown to activate cytokine production via TLR9 (25). We
therefore hypothesized that G3139 administration could enhance
the antitumor effects of f-IgG. Free G3139 or liposomal G3139
have been shown to prolong animal survival. Compared to free
G3139, liposomal G3139 was shown to be significantly more
active in tumor inhibition (Figure 5). Recent studies in our lab
have demonstrated that liposomal G3139 significantly increased
plasma concentration of IFN-γ and IL-6 in mice, and increased
proliferation of the DCs and NK cells (27). In contrast, no down-
regulation of bcl-2 in murine L1210JF cells was observed,
presumably due to sequence mismatches between G3139 and

Folate-IgG for Anticancer Therapy
Bioconjugate Chem., Vol. 21, No. 5, 2010 967
characteristics of a therapeutic antibody. In addition, folate
targeted f-IgG immunotherapy, combined with liposomal G3139,
is a promising strategy for treatment of FR(+) cancers.
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