Cells
NCI-H226 and OVCAR-3 cells were purchased from the American Type
Culture Collection (ATCC). IGROV-1 cells were obtained from the
NCI cell collection. Panc 10.05 cells were previously described
(
23). Panc 3.27 and Panc 3.014
cells were generated in Dr. Elizabeth Jaffee's laboratory. A431-K5 cells
were derived from A431, an epidermoid carcinoma cell line that has
been stably transfected with a plasmid encoding mesothelin (
6). A431-K5-TEM cells were derived
in Dr. Grasso’s laboratory from A431-K5 cells by transfection
with an expression vector containing the full-length endosialin/TEM1 cDNA.
FACS analyses
Cells were harvested in cell dissociation solution (Invitrogen), washed,
and resuspended in ice-cold PBS containing 1% FBS. Primary
antibodies were added at 10 µg/mL and incubated
for 1 hour on ice. Next, the cells were washed with PBS, incubated with
FITC-conjugated goat-anti-human secondary antibody (Southern Biotech,
Birmingham, AL) diluted to 1 µg/mL, washed as
above and analyzed on an EasyCyte Flow Cytometer (Guava Technologies,
Hayward, CA).
Immunohistochemistry
Samples of snap frozen pancreatic cancer were obtained from pancreaticoduodenectomy
specimens or from tissues harvested in accordance with the Gastrointestinal
Cancer Rapid Medical Donation Program at The Johns Hopkins Hospital
(
24). Five micron sections were
cut from each tissue onto sterile glass slides and fixed in 100% ethanol
for 1 minute, followed by rinsing in 1x TBS with Tween
(TBST) for 5 minutes. Next, endogenous peroxidases were blocked
by incubation with 3% hydrogen peroxide for 10 minutes
followed by a second rinse in 1x TBST. Next, slides were
incubated with a 0.5 µg/mL concentration of MORAb-009
antibody (Morphotek Inc., Exton, PA) diluted in 0.2 M Tris-HCL
pH 7.5 for 1 hour at room temperature and again rinsed in 1x TBST
for 5 minutes. Immunolabeling was detected using the LSAB+ Kit
(DAKO) according to the manufacturer's instructions. Slides were counterstained
in hematoxylin for 30 seconds and coverslipped. Staining patterns
were evaluated by two of the authors (S.M. and C.I.D.) as negative
(0), weakly positive (1+), positive (2+) and strongly
positive (3+), and the percent labeling was evaluated on a
10-tiered scale ranging from 10% to 100%.
Internalization of MORAb-009
The human cell lines NCI-H226 and IGROV-1 were cultured in complete
RPMI 1640 medium (1 mM sodium pyruvate, 5 U/mL
penicillin, 5 µg/mL streptomycin, 2 mM
L-glutamine) containing 10% FBS (Invitrogen) at
37˚C, in a humidified atmosphere containing 5% CO2.
One day prior to addition of antibodies, cells were collected by
trypsinization and plated at a density of 500 cells/well
in a 96-well tissue culture plate (Nalgene Nunc) in complete RPMI
1640 medium. MORAb-009, polyclonal, saporin-conjugated goat anti-human
antibodies (Hum-ZAP, Advanced Targeting Systems) or control antibodies were
mixed in various ratios and incubated for 30 minutes at room temperature,
prior to addition to cultured cells. Cells were incubated at 37˚C,
in a humidified atmosphere containing 5% CO2,
for 5 days prior to harvesting. Relative changes in cell numbers
were quantified by the sulforhodamine B method. Briefly, cells were
killed and fixed for one hour on ice by addition of ice-cold 100% trichloroacetic
acid (TCA) directly into the growth medium, to a final concentration
of 10%. TCA was removed by washing fixed cells 5 times
using distilled, deionized water, and allowed to dry in open atmosphere.
100 µL of a solution of 0.4% sulforhodamine B
in distilled, deionized water were added to each well, and incubated
for 10 minutes. The wells were washed 4 times using 1% acetic
acid and allowed to dry in open atmosphere. The resulting precipitate
was dissolved in 100 µL of 10 mM Tris-Cl pH 8.0,
and the absorbance at 540 nm was measured with a microplate
reader. Relative cell numbers were determined by interpolation from
a standard curve constructed by measuring absorbance of known cell
numbers from each cell line, deposited in the bottom of a 96-well
tissue culture plate. Results were expressed as % growth, by
comparison to untreated cells.
ADCC assay
Donor peripheral blood mononuclear cells (PBMCs) used as effector
cells were thawed and kept overnight in Iscove's Modified Dulbecco's
Medium (IMDM) supplemented with 10% fetal calf serum. The
cells were resuspended in medium at a concentration of 107 cells/mL.
The tumor cells used as target cells were detached from the culture
flask and 106 cells in 100 µL fetal
calf serum were labeled with 100 µCi (3.7 MBq) 51Cr
for 2 hours at 37˚C. Alternatively, lysing of cells was
monitored by a standard lactate dehydrogenase (LDH) release assay
following the manufacturer’s instructions (Roche Applied
Science, Indianapolis, IN). Cells were washed three times with 5 mL
of medium and resuspended in medium at a concentration of 105 cells/mL.
Fifty microliters of the tumor cells were seeded in V-bottom 96-well
culture plates. Cells were then incubated with 50 µL medium
containing MORAb-009 or control antibody at concentrations indicated
in the text. After 30 minutes of incubation at 37˚C, 50 µL
of the PBMCs were seeded in V-bottom 96-well culture plates at various
target-effector cell ratios (1:0, 1:25, 1:50 and 1:100) and the
plates were further incubated for 18 hours at 37˚C. The
release of 51Cr in the supernatant was determined in
a gamma counter. Each measurement was carried out in triplicate.
CA125/MUC16-mesothelin interaction assay
OVCAR-3 cells (1 x 105) were seeded
in triplicate in microplates, incubated overnight at 37˚C/5% CO2 and
allowed to form a monolayer. The following day, A431-K5-TEM cells (mesothelin+/TEM1+)
were harvested and loaded with Calcein AM cell dye (Invitrogen Corp.,
Carlsbad, CA) as per the instruction manual. Labeled cells (2 x 105)
were pre-incubated for 1 hour at 4˚C with MORAb-009 or
control antibodies at a final concentration indicated in the figure
legends. Both normal human polyclonal IgG (Jackson Immunoresearch,
WestGrove, PA) and anti-TEM1 antibody (Morphotek Inc., Exton, PA)
were included to control for the specificity of the MORAb-009 inhibition
of the mesothelin-MUC16 interaction. The OVCAR-3 monolayers
were washed once with 200 µL 10% complete RPMI
(cRPMI) and the cells/antibody mixtures added to triplicate
wells for 45 minutes at 4˚C. Wells were gently washed five
times with 200 µL PBS by inverting the plate on paper towels.
Cell adherence and its inhibition by MORAb-009 were quantitated
using the MetaMorph Fluorescence Imaging System (ver.6, Molecular
Dynamics, Downingtown, PA).
In vivo efficacy studies
Gemcitabine and Taxol® were provided by the Division
of Veterinary Resources (NIH). A431-K5 cells were grown in DMEM
with 10% FBS, 2 mM L-glutamine, 1 mM
sodium pyruvate, 100 units/mL penicillin G, 100 µg/mL
streptomycin and 750 µg/mL G418. Tumor experiments
evaluating MORAb-009 in combination with gemcitabine were conducted
using A431-K5 xenografts in nude mice. Four to six week old female athymic
nude mice (National Cancer Institute-Frederick Animal Production
Area, Frederick, MD) were housed in microisolator cages for the
course of the experiment. The research protocol was approved and
the mice were maintained as per institutional guidelines of the
NIH. Two million A431-K5 cells were inoculated subcutaneously into
the right flank of the mice. Tumor dimensions were determined using
calipers and the tumor volume (mm3) was calculated by
the formula: length x (width)2 x 0.4. Treatment was initiated
when tumors reached approximately 50 mm3 in
size. The different treatment regimens included: gemcitabine (80 mg/kg)
alone via i.p. injection on days 7, 9 and 11 after tumor inoculation;
MORAb-009 or Rituximab (50 mg/kg) alone given
i.v. on days 7, 9 and 11 after tumor inoculation; gemcitabine (80 mg/kg)
i.p. on days 7, 9 and 11 in combination with either MORAb-009 or
Rituximab (50 mg/kg) i.v. on days 7, 9, 11, 13,
15 and 17 after tumor inoculation. Mice were sacrificed when the
tumors reached approximately 500 mm3. To test
the in vivo activity of MORAb-009 in combination
with Taxol®, the same A431-K5 tumor xenograft model was
used. On day 4 after tumor cell inoculation when the tumor size
was approximately 60 mm3, mice (n = 7 per
treatment group) were treated with either Taxol® 50 mg/kg i.p.
on day 4, MORAb-009 50 mg/kg i.v. on days 4, 6 and 8 alone or
the combination of Taxol® 50 mg/kg on
day 4 and MORAb-009 50 mg/kg i.v. on days 4, 6,
8, 10, 12 and 14. Mice were sacrificed when the tumors reached approximately
500 mm3.
Non-human primate toxicity study
After preliminary dose-ranging studies, a 23-day intravenous infusion
toxicity study in cynomolgus monkeys with a 28-day recovery period
was conducted under Good Laboratory Practice (GLP). At the saline
control and 15 mg/kg dose levels, three monkeys
per sex were dosed with MORAb-009 twice per week over 23 days (seven
total doses). At the 2 mg/kg dose level, two monkeys
per sex were dosed at the same frequency. Two days after the last
dose, two monkeys per sex were euthanized and their necropsy carried
out. The remaining control and high dose monkeys in the respective
recovery groups were observed for 28 additional days before euthanasia
and necropsy. Clinical evaluations of the monkeys included the following:
physical examinations pre- and post-dosing; daily clinical observations, including
mortality checks; body weight measurements prior to study start,
and twice weekly throughout the study; daily food consumption estimates
beginning one week prior to treatment throughout the study; and
hematology, coagulation, clinical chemistry and urinalysis measurements
prior to study start and at study termination. Postmortem evaluations
included the following: complete macroscopic examinations on all
monkeys; selected organ weights; and complete histopathology examination.
Blood was drawn before and after each dose, as well as during the
recovery period for the determination of the toxicokinetics of MORAb-009
and for the detection of the possible generation of monkey anti-chimeric
antibodies, which were determined by using ELISA.
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