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Immunology |
-Particle Emitting Antibody ([213Bi]J591) for Radioimmunotherapy of Prostate Cancer1
Departments of Pharmacology and Molecular Therapeutics [M. R. M., E. B., D. M., L. L., M. J. C., V. P., M. L., D. A. S.], Radiology [R. D. F.], Pathology [C. C-C.], and Medical Physics [G. S., Å. M. B., W-H. Y.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021; Department of Urology, New York Presbyterian Hospital-Weill Medical College of Cornell University and Ludwig Institute for Cancer Research, New York, New York 10021 [N. H. B.]; Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892 [M. W. B.]; and Pharmactinium, Inc., Chevy Chase, Maryland 20815 [M. W. G.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-particle emitting monoclonal antibody construct targeting
the external domain of prostate-specific membrane antigen (PSMA) was
prepared and evaluated in vitro and in
vivo. The chelating agent,
N-[2-amino-3-(p-isothiocyanatophen-yl)propyl]-trans-cyclohexane-1,2-diamine-N,N',N',N'',N''-pentaacetic
acid, was appended to J591 monoclonal antibody to stably bind the
213Bi radiometal ion. Bismuth-213 is a short-lived
(t1/2 = 46 min) radionuclide that emits high
energy -particles with an effective range of 0.07–0.10 mm that are
ideally suited to treating single-celled neoplasms and micrometastatic
carcinomas. The LNCaP prostate cancer cell line had an estimated
180,000 molecules of PSMA per cell; J591 bound to PSMA with a
3-nM affinity. After binding, the radiolabeled
construct-antigen complex was rapidly internalized into the cell,
carrying the radiometal inside. [213Bi]J591 was
specifically cytotoxic to LNCaP. The LD50 value of
[213Bi]J591 was 220 nCi/ml at a specific activity of 6.4
Ci/g. The potency and specificity of [213Bi]J591 directed
against LNCaP spheroids, an in vitro model for
micrometastatic cancer, also was investigated.
[213Bi]J591 effectively stopped growth of LNCaP spheroids
relative to an equivalent dose of the irrelevant control
[213Bi]HuM195 or unlabeled J591. Cytotoxicity experiments
in vivo were carried out in an athymic nude mouse model
with an i.m. xenograft of LNCaP cells. [213Bi]J591 was
able to significantly improve (P < 0.0031) median tumor-free survival (54 days) in these experiments
relative to treatment with irrelevant control
[213Bi]HuM195 (33 days), or no treatment (31 days).
Prostate-specific antigen (PSA) was also specifically reduced in
treated animals. At day 51, mean PSA values were 104 ng/ml +/- 54
ng/ml (n = 4, untreated animals), 66
ng/ml +/- 16 ng/ml (n = 6, animals
treated with [213Bi]HuM195), and 28 ng/ml +/- 22 ng/ml
(n = 6, animals treated with
[213Bi]J591). The reduction of PSA levels in mice treated
with [213Bi]J591 relative to mice treated with
[213Bi]HuM195 and untreated control animals was
significant with P < 0.007 and
P < 0.0136, respectively. In conclusion,
a novel [213Bi]-radiolabeled J591 has been constructed
that selectively delivers -particles to prostate cancer cells for
potent and specific killing in vitro and in
vivo. |
INTRODUCTION |
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1000 cGy); there was no repeat dosing
due to human antimurine antibody responses, and no patients met
the radiographic or
PSA3
criteria for objective response (4)
. In the second trial,
a high degree of tumor localization was observed in 13 of 15 patients,
but human antimurine antibody response was seen in all patients; some
degree of antitumor effect, as measured by pain relief, was seen in 6
of 10 symptomatic patients. However, once again, no patients met the
radiographic or PSA criteria for objective response (5)
. Several mAbs that target prostate cancer were modified with the N-[2-amino-3-(p-isothiocyanatophenyl)propyl]-trans-cyclohexane-1,2-diamine-N,N',N',N'',N''-pentaacetic acid (SCN-CHXA''-DTPA) moiety, labeled with 213Bi, and their in vitro potency toward cancer cell lines was evaluated (6) . This comparative study of potential therapeutic antiprostate mAbs resulted in the selection of J591 as the IgG to study further. J591 targets the external domain of PSMA (7, 8, 9) . PSMA is a highly restricted prostate epithelial cell integral membrane glycoprotein, providing an immunogenic extracellular domain. PSMA is expressed by a high proportion of prostate carcinomas, and its expression is further increased in higher-grade cancers, in metastatic disease and in hormone-refractory prostate cancers. There are several mAbs targeting the external domain of PSMA that have been developed and described in the literature (7, 8, 9) .
The construction of an -particle emitting IgG radiotherapeutic
moiety represents a "new class" of potent and specific
radiopharmaceuticals used only recently in humans (10
, 11)
. The radionuclide 213Bi is a
short-lived (t1/2 = 46 min)
-particle emitting metal-ion generated from the decay of Ac-225.
Bismuth-labeled HuM195, an anti-CD33 antibody, demonstrated specific
and potent cell killing ability when directed against a leukemia cell
line (12)
. A robust, reliable
225Ac/213Bi generator
system was developed at Memorial Sloan-Kettering Cancer Center
(13
, 14)
and used to treat 18 patients with 80 doses of
[213Bi]HuM195 in a Phase I clinical trial
(11)
. Clinical conditions for reproducible radiolabeling
of antibodies and quality assurance testing were developed
(15)
. Feasibility for human use and therapeutic activity
were demonstrated. The low abundance, low energy 
-emissions
associated with the decay of 213Bi (440 KeV,
frequency of 16% per disintegration) were used in evaluating the
biodistribution patterns of [213Bi]HuM195 in
humans and in performing dosimetry calculations (16, 17, 18)
.
-Particle therapy has been proposed for use in single-cell
disorders, such as leukemias. The purpose of the current study was to
evaluate the biochemistry, biology, and cytotoxicity of a potent
[213Bi]-labeled J591 construct in a series of
in vitro and in vivo models of prostate cancer to
determine whether RIT with -particles also would be suitable for
clinical use against prostate cancer. Such an approach would be
particularly appropriate after debulking radiation or surgery to reduce
the size of metastatic deposits of cancer.
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MATERIALS AND METHODS |
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Conjugation of Bifunctional Chelate to J591.
J591 was conjugated to
N-[2-amino-3-(p-isothiocyanatophenyl)propyl]-trans-cyclohexane-1,2-diamine-N,N',N',N'',N''-pentaacetic
acid (SCN-CHXA''-DTPA), a backbone substituted derivative of DTPA
(19)
, using an Amicon, Inc. (Beverly, MA) continuous
buffer exchange/dialysis apparatus (20)
. The average
number of chelates per antibody ranged from one to three as determined
by the yttrium arsenazo spectrophotometric method (21)
.
Immunoreactivity of the construct was determined as described
(12)
.
Radioisotope Production, Radiolabeling, and Purification of
[213Bi]J591.
Bismuth-213 was produced (22, 23, 24)
from an
225Ac-source generously supplied by the Institute
for Transuranium Elements (Karlsruhe, Germany) or purchased from Oak
Ridge National Laboratory (Oak Ridge, TN). The
225Ac was fashioned into a radionuclide generator
that produced 213Bi using published procedures
(13)
. Radiolabeling and purification of the mAb construct
with 213Bi was carried out using published
methods (12)
. The labeling efficiency of the reaction and
purity of the final product was determined routinely using instant TLC
with silica gel impregnated paper (Gelman Science Inc., Ann Arbor, MI;
Ref. 12
).
Bismuth-213 Activity Determination.
Bismuth-213 activity was measured with a Squibb CRC-17 Radioisotope
Calibrator (or equivalent model; E. R. Squibb and Sons, Inc.,
Princeton, NJ) set at 775 and multiplying the displayed activity value
by 10. The activity value reported by the CRC calibrator was verified
by counting a sample aliquot point source at a fixed geometry on a
pulse height multichannel analyzer (Canberra Industries, Meriden, CT).
The counting efficiency of the 213Bi 440 KeV
-emission was determined using a plot of counting
efficiency-1 versus the -energy of
standard radionuclide sources (13)
.
Binding Affinity and Determination of the Number of
Antigen-binding Sites.
A nonlinear regression curve fit of the data obtained for the specific
bound [111In]J591 was performed to determine
the J591 equilibrium binding affinity with LNCaP cells and the number
of J591-binding sites per cell. Briefly, the
[111In]J591 construct was labeled, purified,
and characterized in a similar fashion to the CHX-A-DTPA-HuM195
construct (12)
. Freshly prepared LNCaP cells at 4°C (5E4
cells/tube) were reacted with a 50-fold excess of unlabeled J591 and
serial increments of [111In]J591(in triplicate)
or just the serial increments of [111In]J591(in
triplicate) to generate the equilibrium J591-binding curves. A
nonlinear regression method was used to fit a curve to the plot of cpm
per cell pellet versus added radiolabeled antibody.
Bmax was corrected for the immunoreactivity of
the preparation and used to determine the number of antigen-binding
sites per cell. An apparent Kd was read from the plot at a value of 0.5
times the immunoreactivity corrected Bmax.
Modulation of Cell Surface Antibody-Antigen Complexes.
Internalization of the cell surface antibody-antigen complex was
determined by incubating 1.0 mg/ml [111In]J591
with 2E5 cells (x2) in a volume of 2.50 ml of RPMI media as a function
of time at 37°C. The [111In]J591 construct
was substituted for the [213Bi]-labeled
construct in this assay because it has a longer half-life and as a
radiometal ion behaves similarly to 213Bi
in vitro; the [111In]J591 construct
was labeled, purified, and characterized in a similar fashion to the
CHX-A-DTPA-HuM195 construct (12)
. Exposed cells were
pelleted at 0.5 min, 2 h, 4 h, and 24 h, and the
pellets were washed twice in RPMI media; the surface-bound
[111In]J591 was stripped with 1 ml of 50
mM glycine (Aldrich Chemical Co., Milwaukee,
WI)/150 mM NaCl (pH 2.8; Aldrich Chemical Co.),
at 24°C for 10 min. Total cell-associated radioactivity and
internalized (acid-resistant) radioactivity were determined by counting
the [111In]-labeled samples with a Packard
Cobra Gamma Counter (Packard Instrument Co., Meriden, CT) with a
15–550 keV energy window. To avoid nonspecific and Fc receptor binding
by the cells, the assays were performed in the presence of 2% human
serum.
[213Bi]J591 Potency and Specificity against Single
Cells in Vitro.
The potency and specificity of [213Bi]J591 for
killing single prostate cancer cells was determined using 5E4 LNCaP
cells (PSMA+) or SKOV3 cells (PSMA-) in 0.200 ml in 96-well plates. In
these experiments, serial dilutions of
[213Bi]J591 were added to the cells to yield
final activity in the wells ranging from 10–10,000 nCi/ml. The
experiments were done with different specific activities of the
bismuth-labeled constructs (0.06–6.4 mCi/mg). The plates were
incubated 24 h at 37°C in 5% CO2. After
incubation, cell viability was determined by
[3
H]thymidine (DuPont NEN, North Billerica, MA)
incorporation (12)
.
[213Bi]J591 Potency and Specificity against
Multicellular Spheroids in Vitro.
The spheroid cytotoxicity experiments used two fixed activity levels
[50,000 nCi/ml (specific activity of 2.5 Ci/g) and 16,500 nCi/ml
(specific activity of 0.6 Ci/g)] of
[213Bi]J591 with treatment of groups of 24
similarly sized spheroids. Controls were spheroids: (a)
exposed to equivalent activity levels and protein concentrations of a
[213Bi]-labeled irrelevant IgG (HuM195);
(b) unlabeled J591; and (c) no treatment (each
group had 24 spheroids). Spheroids of LNCaP cells were initiated using
described methods (25
, 26)
. Initial spheroid volumes were
approximately 1E-12 m3
, which was a spheroid with
a diameter equal to 0.124 mm and comprising about 1000 cells. Spheroid
volumes were measured every 3 days for the duration of the study (1–2
months) using a microscope (Axiophot 2; Carl Zeiss Ltd.,
Göttingen, Germany) with a digital camera (EOS-DCS 5; Kodak,
Rochester, NY; Refs. 26
and 27
).
LNCaP Tumor Model in Mice.
LNCaP tumor cells in Matrigel (Becton Dickinson Labware, Bedford, MA)
were xenografted into athymic nude mice (8 weeks of age). Mice received
an i.m. injection of 6–7E6 LNCaP tumor cells mixed with Matrigel in
the right hind leg at a volume of 0.25 ml. Tumor growth in
vivo was assessed at several early time points by sacrificing mice
and examining the morphology, size, vascularization, and encapsulation
of the tumor cells in the leg histologically.
[213Bi]J591 Potency and Specificity in
Vivo.
Two cytotoxicity experiments were carried out in vivo using
the mouse model described above. In the first experiment, xenografted
mice were separated into two treatment groups (n = 6, each group) and one control group (n = 4) and treated 2 days after tumor implantation as:
(a) cumulative 0.08 mCi [213Bi]J591
(four approximately equal daily doses); (b) cumulative 0.12
mCi nonspecific [213Bi]HuM195 (four
approximately equal daily doses); and (c) no treatment. All
animals were retro-orbitally bled on days 1, 6, and 51 posttreatment,
and PSA levels were assayed using the IMX PSA Assay (Abbott
Laboratories, Abbott Park, IL). Animals were also monitored for the
appearance of a blue-colored lesion beneath the skin at the
site of implantation (indicating the onset of a well-vascularized
tumor) and the rate of tumor growth. In a second study, 32 xenografted
male athymic nude mice were separated into five treatment groups and
one control group and treated 2 days after tumor implantation as:
(a) single 0.09 mCi dose of
[213Bi]J591 (n = 6);
(b) cumulative 0.13 mCi [213Bi]J591
(four approximately equal daily doses; n = 6); (c) single 0.09 mCi dose of nonspecific
[213Bi]HuM195 (n = 5); (d) cumulative 0.12 mCi dose of nonspecific
[213Bi]HuM195 (four approximately equal daily
doses; n = 5); (e) single dose of
0.02 mg of native J591 (n = 5); and
(f) control group receiving no treatment
(n = 5). All animals were retro-orbitally
bled on day 30 posttreatment, and PSA levels were assayed using the IMX
PSA Assay. Animals were also monitored for the appearance of a
blue-colored lesion beneath the skin at the site of
implantation and rate of tumor growth as a function of time. All mice
receiving [213Bi]-labeled mAbs and unlabeled
mAbs were given injections via the retro-orbital sinus while
under anesthesia of xylazine (10 mg/kg) and ketamine*HCl (200 mg/kg),
following the guidelines set forth in institutional protocols. PSA data
were evaluated using an unpaired t test (two-tailed). The
data for median tumor-free survival versus time was
evaluated using a log-rank test and plotted as a Kaplan-Meier survival
curve.
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RESULTS |
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. Using this value of
Bmax, it was estimated that there were 180,000
molecules of J591 bound per cell. The equilibrium binding affinity, Kd,
was taken from the fit curve at 0.5 times Bmax
and estimated to be 3 nM.
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). The total cell-associated counts and, therefore, the cell
surface counts continued to increase as a function of time through
24 h.
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. The LD50 value of
[213Bi]J591 was 220 nCi/ml at a specific
activity of 6.4 Ci/g, 315 nCi/ml at a specific activity of 3.8 Ci/g,
and 4400 nCi/ml at a specific activity approaching 0.06 Ci/g. At the
low specific activities represented by the last group, there is
essentially no specific binding of the -particle emitting J591 to
the targets. Thus, this level of cell kill represents the nonspecific
cytoxicity in the system used here. Additional experiments evaluated
[213Bi]J591 against a PSMA-negative tumor cell
line, SKOV3. The LD50 value derived was 13,000
nCi/ml at a specific activity level of 1.6 Ci/g (data not shown).
Therefore, cytotoxicity was specific activity related, dose related,
and antigen specificity dependent.
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1000
LNCaP cells. The volumes of all of the spheroids treated with a single
dose of [213Bi]J591 decreased markedly over a
2-month period (Fig. 4)
. Their size was more than 100-fold smaller than the untreated spheroid
control group in this time period. The
[213Bi]HuM195-treated spheroids demonstrate a
1-week delay in growth, followed by an increase in volume. Two other
control groups, untreated spheroids and spheroids exposed to unlabeled
J591 (0.027 mg/ml), showed similar increases in volume to each other as
a function of time with an almost 100-fold increase in spheroid volume
after 1 month.
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. At the time RIT was administered (day 2), the tumors were
characterized histologically as disorganized cell clusters and nodules
each comprised of several thousands of cells. The nodules were not
vascularized and not encapsulated. On day 3, the tumors were more
organized and were becoming vascularized, but still not encapsulated.
By the 5th day, vascularization was more pronounced, and on day 7 the
tumors were encapsulated. The purpose of the RIT studies (described
below) was to examine the ability of the agent to treat the tumors
before encapsulation and neovascularization.
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of LNCaP xenografted mice relative to mice treated with
[213Bi]HuM195 or untreated controls. The median
tumor-free survival times were 31 days (n = 4, untreated animals), 33 days (n = 6,
[213Bi]HuM195-treated animals), and 54 days
(n = 6,
[213Bi]J591-treated animals).
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. Mean PSA values, 51 days after treatment, were 104 ng/ml +/- 54
ng/ml (n = 4, untreated animals), 66 ng/ml
+/- 16 ng/ml (n = 6,
[213Bi]HuM195-treated animals), and 28 ng/ml
+/- 22 (n = 6,
[213Bi]J591-treated animals). The reduction of
PSA levels in mice treated with [213Bi]J591
relative to mice treated with [213Bi]HuM195 and
untreated control animals was significant with P < 0.007 and P < 0.0136, respectively.
In another similar experiment where unlabeled J591 was also examined as
an additional control, the mean PSA values 30 days after treatment were
31 ng/ml +/- 20 ng/ml (n = 5, untreated
animals), 36 ng/ml +/- 38 ng/ml (n = 5, 0.02
mg J591-treated animals), 26 ng/ml +/- 21 ng/ml
(n = 10,
[213Bi]HuM195-treated animals), and 12 ng/ml
+/- 8 ng/ml (n = 12,
[213Bi]J591-treated animals) (Fig. 6)
. In this experiment, animals received either one single
213Bi drug administration or four consecutive
daily administrations of a smaller dose of drug. There were no
statistically significant differences in responses (measured PSA
levels) observed between the 1x daily and the 4x daily treatment
regimens for the [213Bi]J591 and the
[213Bi]HuM195 treatments, respectively, nor
between the unlabeled J591 and untreated controls. Reduction of PSA
levels, however, in all mice (n = 12) treated
with [213Bi]J591 (1x daily and the 4x daily
treatment regimens pooled) relative to all mice (n = 10) treated with [213Bi]HuM195 (1x
daily and the 4x daily treatment regimens pooled) and all control
animals (groups untreated and treated with unlabeled J591 pooled;
n = 10) was significant with
P < 0.0443 and P < 0.0192, respectively.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Particle emitting radiolabeled antibody constructs have been
proposed as potent, selective agents to kill single cells (30
, 31)
. We have shown this in vitro and in humans using
an -particle emitting anti-CD33 mAb for treatment of leukemia
(10, 11, 12
, 15 , 16)
. Tens of billions to hundreds of billions
of individual leukemia cells in the blood and marrow were killed
safely. The kinetics and geometry of single-cell killing, however,
might not be predictive of killing micrometastatic clusters of tumor
cells as would be expected with the early spread of carcinomas.
Therefore, we now ask whether an -particle emitting agent would be
useful in a model for solid tumors, such as prostate cancer, that form
micrometastatic disease in the bone marrow. In this study, we describe
for the first time the construction of a prostate-specific,
-particle emitting agent capable of: (a) binding and
internalizing into target cells; (b) selectively killing
both individual cells and 1000-cell spheroid clusters; and
(c) prolonging tumor-free survival and reducing PSA in mice
bearing prostate cancer xenografts.
LNCaP cell kill in vitro using
[213Bi]J591 proved to be both specific activity
dependent and activity concentration dependent. There was a 20-fold
difference in the LD50 values in
vitro, spanning a 100-fold range in specific activities,
indicating the specificity of the drug for cells expressing PSMA.
Additional data demonstrated that [213Bi]J591
did not specifically kill a PSMA-negative tumor cell line, SKOV3,
supporting the fact that the drug does not target or destroy tissue
that does not express PSMA. Therefore,
[213Bi]J591 was an effective and specific
radiolabeled agent for ablation of individual prostate cancer cells
in vitro. This is an advantage of using -particle
emitting radionuclides because the particles transfer their energy in a
region that is in very close proximity to the target.
We have also investigated the potency and specificity of
[213Bi]J591 directed against LNCaP spheroids
initially comprising 1000 cells in vitro. Spheroids of
this size approximate the micrometastatic disease condition and can
serve as a therapeutic test for investigating the potency and
specificity of -particle emitting IgG directed against multicellular
targets. The [213Bi]J591 must be efficacious
against the spheroids if it is expected to be clinically effective
against metastatic prostate cancer. A single dose of the
[213Bi]J591 drug was effective in arresting the
growth of LNCaP spheroid cells relative to an equivalent dose of
[213Bi]HuM195, an irrelevant control mAb that
has a minimal effect against the LNCaP spheroids, a similar dose of
unlabeled J591, and untreated spheroids. Control experiments exhibited
similar spheroid growth kinetics, with the average spheroid volume
increasing almost 100-fold over a period of 1 month. The
[213Bi]HuM195-treated spheroids exhibited a
delay in growth of approximately 1 week, presumably due to nonspecific
irradiation effects. It was estimated that random hits from
particles emitted from nonspecific IgG dispersed in the media could
result in delivery of about 1% of the radiation dose as compared with
a [213Bi]-labeled specific mAb
(27)
. Other
experiments4
have shown that anti-PSMA IgG penetrates one to two cell layers into
spheroids during the time period (45–90 min) that
213Bi would be expected to be most active. The
[213Bi]J591-treated spheroids decreased in
volume by about 10% over a 3-week period with no further growth during
the 2-month duration of the experiment. These data were repeated in a
second experiment with similar results. Because the spheroid model may
resemble the micrometastatic disease situation, it was apparent that
[213Bi]J591, despite incomplete penetration
into the spheroid mass, was able to specifically target large cell
clusters, destroy targeted cells, and interrupt further growth of the
unirradiated spheroid core. Additional studies ongoing in this area are
focusing on the use of a multiple dosing schedule that would
effectively peel away the remaining cell layers that might still be
viable. A multiple dosing schedule may eradicate the disease point
source in its entirety while targeting any other remaining or new
disease sites; however, the schedule and dose details need to be better
understood.
The two therapeutic experiments carried out in vivo
demonstrated the ability of [213Bi]J591 to
improve the duration of tumor-free survival and to suppress PSA
relative to controls in an animal model. Following the trends observed
in the spheroid cytotoxicity experiments, unlabeled J591 was
ineffective in treatment and was comparable with no treatment. In
addition, the [213Bi]HuM195 demonstrated a
minimal effect in delaying tumor growth (2–3 days) and in decreasing
PSA values relative to controls. Table 2
compares the Ps for
the observed PSA values between the
[213Bi]J591-treated mice and the
[213Bi]HuM195-treated mice and controls. The
tumor model used in this study was not optimal for examining the
efficacy of an -particle emitting radioimmunotherapeutic agent
because an -particle has a pathlength of two to four cell diameters.
However, in the absence of reliable metastatic models for prostate
cancer in vivo, we used an i.m. tumor model and treated at a
relatively early time point following tumor inoculation; this allowed
us to observe an effect due to the [213Bi]J591
agent that resulted in tumor growth delay and a lower PSA value
relative to controls. The specific [213Bi]J591
was directed against clusters of cells that at the time of RIT were
approximately the size of the spheroids that were studied in
vitro. As with the spheroids, the tumors in vivo
comprised nodules containing 1000–2000 LNCaP cells that were not
vascularized and not encapsulated.
We have been successful in a Phase I clinical trial using
[213Bi]HuM195 (10
, 11)
to
treat 18 patients with acute myelogenous leukemia. Ten of 12 evaluable
patients had reductions in peripheral blood leukemia cells, and 12 of
18 patients had decreases in bone marrow blasts. Doses up to 1 mCi/kg
were used safely, and there was no acute toxicity seen. In the
treatment of leukemia there may be 1 kg of tumor present (1E12
cells) with 20,000 HuM195 molecules bound by individual HL60
leukemia cells (32)
.
The clinical situation in prostate cancer may favor the use of a
[213Bi]-labeled antibody based on:
(a) low tumor burden; (b) anti-PSMA-IgG-PSMA
internalization; and (c) the relatively large number of
PSMA-binding sites per cell. In comparison with leukemia, there should
be at least 10–100 times less prostate cancer tumor burden at the time
of treatment, even with widespread micrometastatic lesions.
Furthermore, J591 results in the internalization of the J591-PSMA
complex into LNCaP cells (8)
, which favors the therapeutic
use of [213Bi]-labeled IgG constructs because
internalized radiometal ions (a) will tend to remain with
the cell and not be released and carried away from the target and
(b) decays within the cell will necessarily deposit energy
within the cell. We undertook the measurement of the radiometal-labeled
construct-antigen complex internalization to determine the amount of
drug internalized initially and as a function of time. Cellular
internalization of -particle emitting radionuclides insures that the
213Bi particulate decay traverses the cell,
enhancing the cytotoxicity relative to surface-bound species. We
anticipate the ability to internalize much of the targeted, cell bound
[213Bi]J591 activity within a 4-h period
allowing the 213Bi to decay and deliver its dose
efficiently within the target cell. Additionally, it has been observed
that the LNCaP cells continue to bind J591 and internalize it following
exposure to the construct, presumably by expression of new (recycled)
antigen-binding sites (Ref. 8
and this study). From a
therapeutic standpoint, this modulation phenomenon works to therapeutic
advantage because [213Bi]J591 in circulation
can continue to be bound even after initial saturation of the cell
binding sites. The LNCaP cells bind 180,000 molecules of J591, which
was about a 10-fold greater number of binding sites than the number of
CD33 sites per leukemia cell. Assuming that prostate cancer cells
express similar levels of PSMA in vivo, typical therapeutic
specific activities of [213Bi]J591 (10 Ci/g)
would result in delivery of 80 213Bi-atoms per
cell. It has been demonstrated (12)
that several
213Bi-atoms targeted per cell may result in a
lethal dose of particles to half of these cells.
In summary, the radioimmunopharmaceutical described in this study, [213Bi]J591, has been demonstrated to potently and specifically eradicate individual prostate cancer cells and 1000 cell spheroid clusters in vitro and inhibit tumor growth and suppress PSA in vivo in animal models. These features support the investigation of such an agent further in the human clinical setting to treat metastatic prostate cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the CaPCure Foundation and NIH
Grant RO1-CA55349. D. A. S. is a Doris Duke Distinguished Clinical
Science Professor. 
2 To whom requests for reprints should be
addressed, at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue,
New York, NY 10021.
3 The abbreviations used are: PSA,
prostate-specific antigen; mAb, monoclonal antibody; PSMA,
prostate-specific membrane antigen; RIT, radioimmunotherapy; DTPA,
diethylenetriaminepentaacetic acid.
4 G. Sgouros and Å. M. Ballangrud, unpublished
results.
Received 3/ 9/00. Accepted 8/24/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and nonlinear regression curve fit
(solid line) data are presented. Experiments were
conducted as described in "Materials and Methods."
), internalized (
), and
surface bound (
) [111In]J591 as shown for LNCaP cells.
Experiments were conducted as described in "Materials and
Methods."
, 0.06 Ci/g; 
, J591; 
