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Immunology |
Division of Nuclear Medicine, Department of Radiology [P. M. S-J., S. V., S. J. G., C. J. H., D. B.], and Laboratory of Urological Oncology, Department of Urology [V. N., N. H. B.], New York Presbyterian Hospital-Weill Medical College of Cornell University, New York, New York 10021
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSInternalization and Cellular...RESULTSDISCUSSIONREFERENCES |
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Currently, an 111In-labeled form of the 7E11 murine mAb is approved by the Food and Drug Administration (Prostascint) for the clinical detection of recurrent and metastatic prostate cancer in soft tissue (15) . 7E11 mAb binds to the intracellular portion (NH2 terminus) of the PSMA antigen and, as such, does not bind viable cells (13 , 14) . It is believed that successful imaging with Prostascint results from mAb binding to antigen exposed in dead or dying cells within some tumor sites (16 , 17) . Early clinical trials using 90Y-labeled 7E11 resulted in no objective or biochemical (prostate-specific antigen) remissions (7) .
Recently, a series of mAbs to PSMAext has been characterized and reported (13 , 14 , 18) . In this current study, we report on the in vitro evaluation of radiolabeled forms of these antibodies against PSMAext and the selection of interesting candidates for in vivo evaluation of their diagnostic and therapeutic potential.
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MATERIALS AND METHODS |
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LNCaP cells (American Type Culture Collection, Rockville, MD) were grown in RPMI 1640, supplemented with 10% FCS, at a temperature of 37°C in an environment containing 5% CO2. Prior to use, the cells were trypsinized, counted, and suspended in serum-free medium. LNCaP cells were permeabilized by adding methanol at -80°C to the cells. The cells were maintained at -20°C for 20 min before the methanol was removed, and the cells were rehydrated by washing four times with PBS (with 5 mM Ca2+ and 5 mM Mg2+) over 20 min.
Cell membranes were prepared by lysing the cells with a Polytron in a hypotonic buffer [1 mM Na2CO3 (pH 7.4) with 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride). Large fragments were removed by centrifuging at 2000 x g. The supernatant was centrifuged at 150,000 x g for 2 h, and the pelleted membranes were resuspended in PBS, aliquoted, and frozen at -70°C until required.
Radioiodination
The four murine mAbs were radiolabeled with
131I using the Iodogen
(1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril) method
(19)
. Briefly, 10-ml glass tubes were coated with 50 µg
of Iodogen by adding 100 µl of a 0.5 mg/ml solution of Iodogen
(Pierce, Rockford, IL) in chloroform. The chloroform was removed by
blowing a gentle stream of sterile nitrogen into the tube for 30 min
before the tubes were sealed and stored in the dark. The iodination
reaction was initiated by adding between 4 and 40 MBq of
131I (0.01 M NaOH) to 0.08 mg of mAb
in 0.1 ml of ice-cold PBS. This reaction mixture was allowed to react
for 5 min on ice before being loaded onto a 10-ml Biogel-P6 column
(Bio-Rad Laboratories, Hercules, CA) equilibrated with 1% BSA in PBS.
Once the reaction mixture was loaded onto the column, it was washed
with 2 ml of 1% BSA PBS before the main
131I-labeled mAb fraction was eluted with 2 ml of
1% BSA PBS. The amount of free iodine in the
131I-labeled mAb preparations was evaluated using
instant TLC with a silica gel impregnated glass fiber support and a
mobile phase of isotonic saline. Briefly, a portion of the
131I-labeled mAb was spotted on a 10-cm ITLC-SG
strip (Gelman Sciences, Ann Arbor, MI) and developed in isotonic
saline. Once the solvent front had reached the end of the strip, it was
removed from the solvent and cut at an
Rf of 0.5. The two portions were
assayed for radioactivity, and the radiochemical purity determined
using the following equation: radiochemical purity = activity between Rf 0 and 0.5/total
activity in strip.
Antibody Conjugation
J415 and J591 antibodies were modified with DOTA by an analogous
method to that used by Lewis et al. (20)
. This
method uses the direct coupling of one of the four carboxylic acid
groups of DOTA to the primary amines present in the protein structure
(Fig. 1)
. Twenty-five mg of antibody were concentrated in a
Mr 30,000 Microsep centrifugal
concentrator (Pall Filtron, Northborough, MA) and washed with
5 x 4 ml of 1% DTPA (pH 5.0) over a period of 24 h. The antibody buffer was then changed to 0.1 M
phosphate (pH 7.0) using the same centrifugal technique. An active
ester of DOTA was created by dissolving 146 mg of DOTA (0.361 mmol) and
36 mg of N-hydroxysuccinimide (0.313 mmol) in 2 ml of water
and adjusting the pH to 7.3 with NaOH, prior to the addition of 10 mg
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. This reaction mixture
was cooled on ice for 1 h before being added to the J591 solution.
The resultant DOTA-antibody conjugate was separated from the excess
DOTA and other reactants by repeated washing with 0.3
M NH4OAc (20 x 4 ml) and centrifugal concentration.
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111In and 90Y Labeling of DOTA Conjugate
Radiolabeling of DOTA-J591 with 111In was
achieved by adding the radionuclide (in dilute HCl) to the ammonium
acetate-buffered DOTA-J591. Briefly, a mixture composed of 20 µl of
111InCl3 (300 MBq), 0.01
M HCl, and 400 µl of DOTA-J591 (4 mg/ml; 0.3
M NH4OAc, pH 7) was allowed to react
at 37°C for 20 min. The reaction mixture was then separated on a
20-ml Biogel-P6 column equilibrated with 4 x 10 ml of
sterile 1% HSA in PBS. After the reaction mixture was loaded onto the
column, it was washed with an additional 5 ml of 1% HSA PBS before the
main 111In-DOTA-J591 fraction was eluted with 3
ml of 1% HSA PBS. A similar procedure was used for radiolabeling with
90Y, but an incubation time of 5 min was used,
and the labeling mixture included 50 mM ascorbic acid.
Free 111In in the radiolabeled DOTA-J591 preparations was determined using the ITLC method with a silica gel-impregnated glass fiber support and a mobile phase of 1% DTPA (pH 5.5). A portion of the radiolabeled DOTA-J591 was spotted on a 10-cm ITLC-SG strip and developed in 1% DTPA (pH 5.5). Once the solvent front had reached the end of the strip, it was removed from the solvent and cut at a Rf of 0.5. The two portions were assayed for radioactivity, and the radiochemical purity was determined using the equation described earlier.
Chelate Stability Studies
111In-labeled DOTA-J591 and DTPA-7E11 were
mixed with an equal volume of 50 mM DTPA and maintained at
37°C. Periodically, samples were removed and spotted on a 10-cm
ITLC-SG strip and developed in 0.9% NaCl. Once the solvent front had
reached the end of the strip, it was removed from the solvent and cut
at a Rf of 0.5. The two portions were
assayed for radioactivity, and the amount of intact chelate was
determined using the equation described earlier.
Binding Studies
Immunoreactivity.
The immunoreactivity of the 131I- and
111In-labeled mAb preparations was assessed by
the method of Lindmo et al. (21)
, which
extrapolates the binding of the radiolabeled antibody at an infinite
excess antigen. Briefly, six test solutions were prepared (in
duplicate) and contained 20,000 cpm of the radioiodinated antibody, and
increasing amounts of membranes were prepared from LNCaP cells in a
total test volume of 250 µl of PBS (0.2% BSA, pH 7.4). The solutions
were incubated at 37°C for 45 min prior to being filtered through a
glass membrane filter and washed with ice-cold 10
mM Tris-0.9% NaCl buffer. Filters were counted
in a gamma counter with standards representing the total radioactivity
added. Data were then plotted as the reciprocal of the substrate
concentration (X axis) against the reciprocal of the fraction bound (Y
axis). The data were then fitted according to a least squares linear
regression method using Origin software (Microcal Software, Inc.,
Northampton, MA). The Y intercept gave the reciprocal of the
immunoreactive fraction. A similar method using intact or permeated
LNCaP cells and centrifugational isolation of the cells gave the same
results.
Competitive Binding Studies.
Competitive binding studies were performed with each of the
radioiodinated antibodies and the four unlabeled antibodies using
either LNCaP tumor sections or membranes derived from LNCaP tumors.
Acetone fixed and frozen 10-µm tumor sections were soaked in Tris
buffer [170 mM (pH 7.4), with 2 mM
CaCl2 and 5 mM KCl] for 15 min and
then washed with Tris buffer (170 mM, pH 7.4). The sections
were then incubated with the radioiodinated antibodies in the presence
of 100 nM concentrations of each of the unmodified mAbs for
1 h at 4°C. Sections were washed three times with PBS (0.2%
BSA) and once with Tris buffer (170 mM, pH 7.4) prior to
being fixed with acetone and exposed with BioMax film (Kodak). The
assay using the membranes typically used 50 µg of membranes, 10 fmol
of iodinated antibody, and amounts of competing antibody from 0.25 fmol
to 25 pmol in a 250-µl volume of PBS (0.2% BSA). Membranes were
isolated as described above, and data were analyzed by a least squares
regression method and Origin software (Microcal Software, Inc.) was
used to determine the IC50s.
Saturation Binding Studies.
Saturation binding studies were performed with each of the radiolabeled
antibodies using substrates of intact and permeated LNCaP cells.
Briefly, 10 test solutions were prepared (in duplicate) and they
contained increasing amounts of the radioiodinated antibodies, 500,000
LNCaP cells in a total volume of 250 µl of PBS (0.2% BSA, pH 7.4).
The solutions were incubated at 4°C for 1 h and centrifuged and
washed twice with ice-cold PBS (0.2% BSA). For each concentration of
radiolabeled antibody, nonspecific binding was determined in the
presence of 100 nM of the unmodified antibody. The data
were analyzed with a least squares regression method (Origin; Microcal
Software, Inc.) to determine the Kd
and Bmax values, and a Scatchard
transformation was performed.
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Internalization and Cellular Processing of J415 and J591 |
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0.1–0.2 µg) were added to cells and
allowed to incubate for 1 h. The medium was then removed, and the
cells were washed once with fresh media. One ml of fresh medium was
added, and the cells were incubated for up to 2 days at 37°C.
Triplicate samples were periodically removed, and the medium was
isolated. Surface bound activity was stripped and collected with an
ice-cold acid wash (100 mM acetic acid, 100 mM
glycine, pH 3.0). The cells were then treated with 1 ml of a 1%
solution of Triton X-100 (containing 5 µg/ml each of antipain,
pepstatin, and leupeptin as well as 1 mM
phenylmethylsulfonyl fluoride) and kept at on ice for 20 min. The
resultant suspension was then centrifuged, and the three samples were
counted with a gamma counter. The medium and supernatants were also
analyzed by ITLC and size exclusion HPLC to determine the amounts of
free iodide produced or the size of the radioactive species created. |
RESULTS |
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). This method gave immunoreactivities of >75% for all mAbs tested.
When labeling conditions were increased to produce specific activities
>350 MBq/mg, the immunoreactivity was compromised.
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111In-Chelate Stability Studies.
A direct comparison of the chelate stability of
111In-DTPA-7E11 and
111In-DOTA-J591 showed that
111In was lost from DTPA-7E11 with an apparent
half-life of 11 h, whereas the DOTA chelate had an apparent
half-life exceeding 1000 h (Fig. 3)
.
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. The J415 mAb had an mean IC50 of
1.5 nM (±0.9; n = 6),
and J591 had a mean IC50 of 6.6
nM (±4.5; n = 6).
Similarly, 131I-labeled J533 could be displaced
by J533 and J591 but not by J415 (Fig. 4B)
. In these
studies, J533 had a mean IC50 of 2.3
nM (±1.5; n = 3), and
J591 had a mean IC50 of 1.7
nM (±1.3; n = 3).
Finally, 131I-labeled J591 could be displaced by
J415, J533, and J591 (Fig. 4C)
. The observed
IC50s were 1.3 nM (±0.9;
n = 6) for J415, 7.7 nM
(±5.5; n = 6) for J533, and 3.1
nM (±1.5; n = 6) for
J591. The 7E11 mAb did not inhibit the binding of J415, J533, or J591
(or vice versa). These data are consistent with earlier data
(11)
that J415, J533, or J591 bind to the extracellular
domain of PSMA, whereas 7E11 binds to the intracellular domain of PSMA
(13)
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, demonstrated that J415 and J591 bound with a similar affinity
(Kds 1.76 ± 0.69 and
1.83 ± 1.21 nM), whereas J533 had
a lower affinity (Kd 18 ± 5
nM). In parallel studies, all four mAbs bound to
a similar number of PSMA sites expressed by permeabilized cells
(1,000,000–1,300,000 sites/cell). In parallel studies performed with
viable LNCaP cells, J415, J533, and J591 bound to a similar number of
PSMA sites (i.e., 600,000–800,000 sites/cell). In contrast,
7E11 specifically bound to only 10–15% of the PSMA sites expressed by
apparently intact LNCaP Cells (Kd,
6.69 nM); but when the cells were deliberately
ruptured (Fig. 6)
, 7E11 bound to a similar number of antigen sites as the other three
mAbs. In parallel studies, using 131I-labeled
J591, permeabilized cells expressed about twice the amount of PSMA as
intact LNCaP cells, suggesting that not all available PSMA is
simultaneously expressed on the cell surface.
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. For both mAbs, a biexponential curve fit of the data showed that
10% of the radioactivity was released from the cells with an
apparent half-life of 1 h, and the remaining 90% was released
into the medium with apparent half-lives of 31 and 38 h for J415
and J591, respectively. In parallel studies,
131I-labeled J415 consistently showed a faster
release of radioactivity than 131I-labeled J591.
Little or no activity (<1%) was associated with the Triton X-100 (or
NaOH) insoluble cell pellet. Analysis of the Triton X-100 soluble
fractions indicated that there were no appreciable amounts of free
131I present (<1%). HPLC and TLC analysis of
the culture medium showed that a large iodinated species, which
corresponded to the same size as the intact mAbs, was being released
from the cells, but this never amounted to >10% of the total
activity, and after 4–6 h, no further release of this radioactive
species was observed. The predominant metabolite of the iodinated mAbs
found in the cell medium had the same HPLC and TLC elution profile as
free 131I-. Several
studies compared 131I-labeled J591 and DOTA-J591,
and no significant differences in the retention of
131I by the cells were noted between the two
forms of the same mAb. In all of the studies performed, no increase in
cell death was noted as compared with the control groups that received
no radiolabeled antibodies.
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. For
111In-DOTA-J415, a biexponential curve fit of the
data showed that 20% of the radioactivity was released from the
cells with an apparent half-life of 2 h, and the remaining 80%
was released into the medium with an apparent half-life of 160 h.
For 111In-DOTA-J591, the cellular release of
111In species was much slower, and a
biexponential curve fit of the data showed that about 5–10% of the
radioactivity was released from the cells with an apparent half-life of
1 h, and the remaining 90–95% was being released into the medium
with an apparent half-life of 520 h. Little or no activity (<1%)
was associated with the Triton X-100 (or NaOH) insoluble cell pellet.
HPLC and TLC analysis of the cell medium showed that a large
111In species, which corresponded to the same
size at the intact mAbs, was being released from the cells, but this
never amounted to >10% of the total activity, and after 4–6 h, no
further release of this radioactive species was observed. For both J415
and J591, two main 111In-labeled metabolites were
observed in the medium. Analysis of the cell-associated radioactivity
(Fig. 8)
demonstrated the rapid formation of two groups of metabolites (based
on molecular size). One group of metabolites achieved a maximum
concentration after 1–2 h, after which it began to steadily decline.
The second group of metabolites, however, demonstrated an
ever-increasing intracellular concentration. This second metabolite did
not behave the same as
111In3+ (HPLC or TLC), but
rather it had a similar molecular weight as an
111In-DOTA or an
111In-DOTA-amino acid fragment. The first
metabolite had a molecular weight between that of the intact mAb and
the second metabolite (Mr
10,000–30,000).
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. However, by 4 h after the addition of the radioactivity, the
cells treated with 111In-DOTA-J591 have
incorporated and retained more activity than those treated with
111In-DOTA-J415.
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DISCUSSION |
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Early approaches to labeling mAbs with radiometals used DTPA, which in its dicyclic anhydride form could be conveniently coupled to mAbs (22) . Unfortunately, this simple coupling chemistry produced a more labile chelate than bifunctional forms of the same unconjugated DTPA chelator (23) . Macrocylic chelators have shown even higher kinetic stability (24) , but they are even more time consuming to chemically synthesize (25) . DOTA can be coupled directly to mAbs using simple chemistry and commercially available materials (20) .
The reaction kinetics for 111In and DOTA are longer than for DTPA, but an incubation period of 15 min can give high labeling yields. The DOTA chelator was immensely superior to DTPA in its ability to tightly chelate 111In in the presence of an excess of competing ligand. This is in agreement with other studies (20 , 26) and underlies the importance of using stable chelates with mAbs that can stay in circulation for prolonged periods of time in the presence of competing ligands (e.g., transferrin). The higher stability of the 111In-labeled DOTA complex relative to the 111In-labeled DTPA complex also applies for the 90Y complex (20 , 26) and is an important prerequisite for radiolabeled mAbs used for either diagnosis or therapy because optimal tumor:nontumor ratios are often achieved after 2–4 days. Because 111In-DOTA-J591 is stable to DTPA competition, it enables nonspecifically bound 111In to be removed by challenging with DTPA and a simple column separation to yield a highly pure radiopharmaceutical.
High binding affinity between the mAb and the target antigen is another prerequisite to in vivo targeting of tumor antigens. The binding studies with the iodinated mAbs showed that two of three of these mAbs against PSMAext (i.e., J415 and J591) and 7E11 have similar nanomolar binding affinities. The use of intact and ruptured cells showed clearly that 131I-labeled 7E11 binds to the intracellular domain of PMSA, consistent with other reports (13 , 17) . There was some binding of 7E11 to "intact" LNCaP cells, but that could be explained by the presence of a small population of cells ruptured during the trypsinization of the cells from the cell culture flasks and subsequent handling during resuspension. Because J591 recognized and specifically bound to twice the number of PSMA sites in permeabilized cells as opposed to intact cells, this suggests that only 50% of all cellular PSMA is exposed extracellularly. Also, 10–15% binding of 7E11 could be explained by the presence of a population of 5–7% of permeabilized cell in the "intact" cell preparation. This was confirmed in other studies that examined the binding and cellular uptake of the mAbs with plated LNCaP cells and showed a 7E11 uptake of 3–4% that of J415 and J591. This quantitative difference seen might explain why one group claims that the 7E11 binds to apparently intact LNCaP cells (27) , whereas other groups report no such binding (13 , 17 , 18) . Additionally, the Barren study (27) used cells that were scrapped from the monolayer, a procedure known to create significant cell rupture.
Effective systemic targeting of tumors has been achieved with both iodinated and metallochelated antibodies. A significant factor in selecting between the two approaches is in whether the antibody is internalized. In the case of an internalizing antibody, directly iodinated antibodies are metabolized within the cell, and the main metabolites of iodide and iodotyrosine are then freely released from the cell. Conversely, the metal chelate-labeled antibody is metabolized to leave a chelate-amino acid fragment that is typically not released from the cell and is trapped for further degradation. This effect can produce vastly different residence times for two differently labeled forms of the same antibody (28) . In the LNCaP cell model, PSMA is known to be an internalizing surface bound glycoprotein (18) . These studies clearly support the notion that metallolabeled mAbs are superior for cells expressing internalizable PSMA and that metabolites from the DOTA-mAb conjugate are not appreciably released from LNCaP cells.
In conclusion, both J415 and J591 have similar nanomolar affinities to PSMA as 7E11. Similarly, these two mAbs are far more readily bound and were internalized by live LNCaP cells than 7E11. The 111In-labeled DOTA conjugates are able to associate more radioactivity with LNCaP cells than the comparable iodinated forms. The 111In-labeled DOTA conjugates are also more stable to loss of 111In than DTPA-7E11. These findings make DOTA-J415 and DOTA-J591 attractive candidates for further evaluation as either diagnostic or radiotherapeutic agents in patients with various cancers that express PMSA.
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FOOTNOTES |
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1 This work was supported by Grant PC970229 from
the United States Department of Army and grants from Yablans Research
Fund and CaP Cure. N. H. B. is a consultant to BZL Biologics, Inc.
The agreement that N. H. B. has with BZL is managed by Cornell
University in accordance with its conflict of interest policies. 
2 To whom requests for reprints should be
addressed, at Weill Medical College of Cornell University, 525 East
68th Street, Starr 221, New York, NY 10021. E-mail: psj2001{at}med.cornell.edu
3 The abbreviations used are: PCa, prostate
cancer; mAb, monoclonal antibody; PSMA, prostate-specific membrane
antigen; DOTA,
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic
acid; BSA, bovine serum albumin; TLC, instant TLC; HPLC,
high-performance liquid chromatography; DTPA,
diethylenetriaminepentaacetic acid.
Received 5/ 3/00. Accepted 7/17/00.
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M. D. Henry, S. Wen, M. D. Silva, S. Chandra, M. Milton, and P. J. Worland A Prostate-Specific Membrane Antigen-Targeted Monoclonal Antibody-Chemotherapeutic Conjugate Designed for the Treatment of Prostate Cancer Cancer Res., November 1, 2004; 64(21): 7995 - 8001. [Abstract] [Full Text] [PDF]
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M. I. Milowsky, D. M. Nanus, L. Kostakoglu, S. Vallabhajosula, S. J. Goldsmith, and N. H. Bander Phase I Trial of Yttrium-90--Labeled Anti--Prostate-Specific Membrane Antigen Monoclonal Antibody J591 for Androgen-Independent Prostate Cancer J. Clin. Oncol., July 1, 2004; 22(13): 2522 - 2531. [Abstract] [Full Text] [PDF]
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P. M. Smith-Jones, S. Vallabhajosula, V. Navarro, D. Bastidas, S. J. Goldsmith, and N. H. Bander Radiolabeled Monoclonal Antibodies Specific to the Extracellular Domain of Prostate-Specific Membrane Antigen: Preclinical Studies in Nude Mice Bearing LNCaP Human Prostate Tumor J. Nucl. Med., April 1, 2003; 44(4): 610 - 617. [Abstract] [Full Text] [PDF]
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S. E. Lupold, B. J. Hicke, Y. Lin, and D. S. Coffey Identification and Characterization of Nuclease-stabilized RNA Molecules That Bind Human Prostate Cancer Cells via the Prostate-specific Membrane Antigen Cancer Res., July 15, 2002; 62(14): 4029 - 4033. [Abstract] [Full Text] [PDF]
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 M. R. McDevitt, D. Ma, L. T. Lai, J. Simon, P. Borchardt, R. K. Frank, K. Wu, V. Pellegrini, M. J. Curcio, M. Miederer, et al. Tumor Therapy with Targeted Atomic Nanogenerators Science, November 16, 2001; 294(5546): 1537 - 1540. [Abstract] [Full Text] [PDF]
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 D. C. Saffran, A. B. Raitano, R. S. Hubert, O. N. Witte, R. E. Reiter, and A. Jakobovits Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts PNAS, February 8, 2001; (2001) 51624698. [Abstract] [Full Text]
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D. C. Saffran, A. B. Raitano, R. S. Hubert, O. N. Witte, R. E. Reiter, and A. Jakobovits Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts PNAS, February 27, 2001; 98(5): 2658 - 2663. [Abstract] [Full Text] [PDF]
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) or LNCaP cell membranes (•), gives the same
immunoreactivity of 
), J533 (•), or J591 (
). After 1 h at
37°C, the medium was removed, and the cells were washed once with
fresh medium. One ml of fresh medium was added, and the cells were
incubated for up to 2 days at 37°C. At various time points, the
location and form of the radioactivity was determined.
Bars, SD.
