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Two-Step Targeting of Xenografted Colon
Carcinoma Using a Bispecific Antibody and
188Re-Labeled Bivalent Hapten: Biodistribution
and Dosimetry Studies
Jean F. Gestin, Anthony Loussouarn, Manuel Bardiès, Emmanuel Gautherot, Anne Gruaz-Guyon,
Catherine Saı̈-Maurel, Jacques Barbet, Chantal Curtet, Jean F. Chatal, and Alain Faivre-Chauvet
Institut de Biologie, Institut National de la Santé et de la Recherche Médicale, Nantes; Hopital St. Antoine, Institut National de la
Santé et de la Recherche Médicale, Paris; and Immunotech, Marseille, France
Radioimmunotherapy (RIT) is currently being considered for the
treatment of solid tumors. Although results have been encouraging
for pretargeted 131I RIT with the affinity enhancement system (AES),
the radionuclide used is not optimal because of its long half-life,
strong g emission, poor specific activity, and low b particle energy.
188Re, though unsuitable for direct antibody labeling, could be used
with the AES two-step targeting technique. The purpose of this
study was to compare the distribution and dosimetry of a bivalent
hapten labeled with 188Re or 125I. For dosimetry calculations and
biodistribution data, 125I was substituted for 131I. Methods: After
preliminary injection of a bispecific anticarcinoembryonic antigen
(CEA) or antihapten antibody (Bs-mAb F6-679), AG 8.1 or AG 8.0
hapten radiolabeled with 188Re or 125I was injected into a nude
mouse model grafted subcutaneously with a human colon carcinoma cell line (LS-174-T) expressing CEA. A dosimetry study was
performed for each animal from the concentration of radioactivity
in tumor and different tissues. Results: Radiolabeling of AG 8.1
with 125I afforded a 40% yield with a specific activity of 11.1
MBq/nmol after purification. Radiolabeling of AG 8.0 with 188Re
afforded a 72% yield with a specific activity of 31.82 MBq/nmol. In
all experiments, the percentage of tumor uptake of 125I-AG 8.1 was
always significantly greater than that of 188Re-AG 8.0. The corresponding tumor-to-tissue ratios reflected uptake values. The least
favorable tumor-to-normal tissue ratios in the dosimetry study were
8.1 and 8.5 for 131I (tumor-to-blood ratio and tumor-to-kidney ratio,
respectively) and 2.3 for 188Re (tumor-to-intestine ratio). Conclusion:
This study indicates that 188Re can be used for radiolabeling of hapten
in two-step radioimmunotherapy protocols with the AES technique.
188Re has a greater range than 131I, which should allow the treatment
of solid tumors around 1 cm in diameter. Although the method used
for hapten radiolabeling did not provide optimal tumor uptake, the use
of a bifunctional chelating agent associated with AG 8.1 should solve
this problem.
Key Words:
pretargeting
188Re;
radioimmunotherapy; bispecific antibody;
J Nucl Med 2001; 42:146 –153
Received Mar. 15, 2000; revision accepted Jun. 20, 2000.
For correspondence or reprints contact: Jean F. Gestin, PhD, Institut de
Biologie, Unit 463, Institut National de la Santé et de la Recherche Médicale,
9 Quai Moncousu, 44093 Nantes cedex, France.
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R
adioimmunotherapy (RIT) has proven to be efficient
for the treatment of low-grade lymphomas, but results for
solid (generally bulky) tumors have been disappointing
(1– 4) because of the low percentage of tumor uptake
achieved (,0.01%) and the relatively high nonspecific uptake with the directly labeled monoclonal antibodies (in
whole immunoglobulin G or fragment form) used in most
studies. Two-step targeting techniques, which substantially
reduce the uptake of activity in normal tissues, should help
overcome these disadvantages (5). Several preclinical and
clinical studies have shown the benefit of using the affinity
enhancement system (AES), which associates a bispecific
antibody with a bivalent hapten (Fig. 1). The haptens used
have been radiolabeled first with 111In for diagnostic studies
and then with 131I for RIT. The mean energy of b2 particles
emitted by 131I limits the efficacy of this radionuclide to
small tumor targets. For larger targets, radionuclides emitting more energetic b2 particles seem to be preferable (6).
Among these isotopes, 188Re is a good candidate because of
its physical characteristics (half-life, 16.98 h; b2, 2.118 and
1.962 MeV) and production mode (188W/188Re generator)
(7–10). However, the possibility of using this radionuclide
with a directly radiolabeled antibody for RIT is limited by
its short physical half-life and the specific activities obtained during direct radiolabeling of antibodies (70 – 80
MBq/nmol) (7–9) as well as slow tumor uptake (11,12). The
AES two-step targeting technique provides rapid tumor
uptake because of the small size and hydrophilic properties
of the bivalent hapten and its long retention time at the
tumor site attributed to specific binding to prelocalized
antibodies (5,13–15).
The purpose of this study was to compare the distribution
and dosimetry of a bivalent hapten labeled with 188Re or 125I
after preliminary injection of a bispecific anticarcinoembryonic antigen (CEA) or antihapten antibody into a nude
mouse model grafted subcutaneously with a human colon
carcinoma cell line (LS-174-T) expressing CEA.
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FIGURE 1.
MATERIALS AND METHODS
The bispecific antibody (Bs-mAb F6 – 679) used in this study
was composed of an F(ab9) fragment of an antiCEA antibody (F6)
coupled chemically to an F(ab9) fragment of an antiglycylsuccinimidylhistamine antibody (679). The bispecific antibody was supplied by Immunotech (Marseille, France) as a 3 mg/mL solution in
0.1 mol/L phosphate buffer and 0.05 mol/L EDTA, pH 7.
The hapten used, AG 8.1, was an octapeptide grafted with two
glycylsuccinimidylhistamine residues in « of lysines 1 and 3. Its
semideveloped formula is shown in Figure 2. AG 8.1 may be
readily radiolabeled with radioactive iodine.
For 188Re labeling, the peptide was derivatized with an Sacetylthioacetyl residue at the N-terminal position. The new derivative thus obtained was designated as AG 8.0 (16). The two
haptens (AG 8.1 and AG 8.0) were supplied by Immunotech as an
aqueous solution at 1 mg/mL.
AES.
Hapten Radiolabeling
125I-AG 8.1. AG 8.1 was labeled with 125I using the chloramine-T technique (17). After labeling, the hapten was purified
using a SepPak C18 cartridge (Millipore, Saint Quentin Yvelines,
France) by successive injections of the labeling solution (5 mL
0.05% trifluoroacetic acid in water [Aldrich, Saint Quentin Fallavier, France] and then 5 mL of a 3:2 mixture of 0.1 mol/L
phosphate buffer solution, pH 7, and ethanol). Under these conditions, free 125I was eluted in the acid aqueous phase and the
radiolabeled hapten was eluted in the first 2 mL of the ethanol
phase. The radiochemical purity of the purified solution was measured by thin-layer silica gel chromatography (Kieselgel 60 F254;
Merck, Nogent sur Marne, France) using methanol as the migration solvent. The immunoreactive fraction was measured by determining the percentage of activity binding to tubes coated with
the antihistamine antibody 679. The chromatograms were analyzed
FIGURE 2. Plane semideveloped formula of AG 8.1 (A) and AG 8.0 (B).
BISPECIFIC MONOCLONAL ANTIBODY
AND 188RE-LABELED
HAPTEN • Gestin et al.
147
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after exposure on a phosphorus screen (Molecular Dynamics,
Sunnyvale, CA) using IPLab-Gel software (Analytics Corp., Atlanta, GA).
188Re-AG 8.0. AG 8.0 was labeled with 188Re by ligand exchange according to a method derived from that of Joiris et al.
(18). Briefly, 188Re-glucoheptonate was obtained quantitatively by
addition under a nitrogen atmosphere of 740 MBq (20 mCi; 3 mL)
sodium perrhenate in a glass flask containing 150 mg sodium
glucoheptonate (Aldrich), 8.4 mg sodium hydrogenocarbonate
(Aldrich), and 1.25 mg stannous chloride (Aldrich). The solution
was incubated for 1 h at room temperature and controlled by
instant thin-layer chromatography on silica gel (ITLC-SG;
Gelman-OSI, Elancourt, France) using 0.9% sodium chloride and
acetone as migration solvents. During the incubation period, AG
8.0 was deacetylated by addition of excess sodium hydroxide (1N).
A spot test with dithionitrobenzoic acid was performed on an
aliquot of the deprotected solution to check for the presence of free
thiol (19). Twenty-one nanomoles of deprotected AG 8.0 were
added to the 188Re-glucoheptonate solution, which was then incubated for 15 min at 100°C before purification, and the control
solution under the same conditions as those during radiolabeling of
AG 8.1 by 125I.
Animal Studies
For each radionuclide, 15 nude mice (Swiss/nu/nu; Iffa-Credo,
L’Arbresle, France) were used (30 animals total). Each animal was
xenografted subcutaneously in the right flank with 1 million cells
of the LS-174-T colon cancer cell line (American Type Culture
Collection, Rockville, MD), which strongly expresses CEA. Two
weeks after grafting, the tumors measured 50 –500 mm3. Each
mouse was injected intravenously in a tail vein with 50 mg (0.5
nmol/100 mL) Bs-mAb F6-679. On the basis of data obtained from
other studies (20 –25), a second injection was performed (24 h after
this first injection) with either 0.25 nmol 125I-AG 8.1 (11.1 MBq/
nmol) or 0.25 nmol 188Re-AG 8.0 (31.8 MBq/nmol).
Biodistribution Study
For each radionuclide, a group of 3 animals was killed at 5 min
and at 1, 5, 24, and 48 h after injection of the radiolabeled hapten.
For each time point, the main tissues and tumors were removed,
dried, weighed, and counted in a g scintillator (Compugamma;
LKB-Pharmacia, Uppsala, Sweden) in parallel with a calibrated
radioactive decay standard. The results are expressed as a percentage of the injected dose per gram (%ID/g) of tissue and as
tumor-to-tissue ratios.
dosimetry instead of 125I for several reasons: cost, radioprotection,
and identical biodistributions. The counting and calculation of the
percentage of the injected dose per animal tissue was determined
simultaneously with that of a weighed standard aliquot of the
injected dose. To minimize counting errors associated with radiation self-absorption by tissues, the same weight of each organ
sample was measured. The mean value of this concentration at
each time point allowed curves to be plotted according to a
theoretic one-compartment extravascular model. The results
(initial concentration, C0) were used, after correction of the physical half-life, to calculate the mean dose delivered by a theoretic injection of 3.7 MBq 131I or 188Re according to a MIRD
formula (26):
# ~target 4 source! 5 Ã source 3
D
D 3 f ~target 4 source!
,
m target
# (target 4 source) is the mean absorbed dose (in Gy)
where D
delivered to the target by the source, Ãsource is the cumulated
activity in the source (in Bq 3 s), D (in J/(Bq 3 s)) is the sum of
energies emitted by desintegration, f(target 4 source) is the
fraction of energy emitted by the source that is absorbed by the
target, and mcible is the mass of the target.
To simplify the calculations, it was considered in a first approximation that the sources and targets were identical, and the emissions were regarded as nonpenetrating. In this case,
# ~source 4 source! 5 Ã source 3
D
D 3 f ~source 4 source!
m source
becomes:
# organ 5 Ã organ 3
D
D
5 C̃ organ 3 D,
m organ
which assumes that the emitted particles deposit their energy
locally:
f ~source 4 source! 5 1.
Calculations were performed with the tabulated D (27) for
and 188Re, which gave respectively:
D 5 1.24 3 10 213 ~kg 3 Gy!/~Bq 3 s! for
188
131I
Re
and
D 5 3.04 3 10 214 ~kg 3 Gy!/~Bq 3 s! for
131
I.
188Re-AG
8.0 Stability
An analysis of the immunoreactive fraction of blood and urine
was also performed using the same procedure as that used for the
radiolabeling control. Statistical analysis of the results was done
by ANOVA using Statview II software (Abacus Concepts, Berkeley, CA).
Measurements of immunoreactive fractions were performed at 5
min and at 1, 5, 24, and 48 h in blood, urine, and the 188Re-labeled
hapten solution after purification. This control solution was kept at
room temperature throughout the experiment, with a starting activity of 185 MBq/mL (5 mCi/mL). Measurements were performed
to study the instability of the radiolabeled hapten.
Dosimetry Study
For each mouse, the concentration of radioactivity (kBq/g) was
calculated in different tissues or tumors. 131I was considered for
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RESULTS
Hapten Radiolabeling
125I-AG 8.1. During radiolabeling of AG 8.1 with 125I, a
40% yield was obtained after addition of 259 MBq 125I to 11
nmol AG 8.1. The specific activity of 125I-AG 8.1 after
purification was 11.1 MBq/nmol, with a radiochemical purity of 100% by chromatography. The immunoreactive fraction was measured to 96.4%.
188Re-AG 8.0. The radiochemical purity of the 188Reglucoheptonate solution used in the radiolabeling of AG 8.0
was 80% by chromatography. The impurities in the preparation represented 12% reduced and hydrolyzed rhenium
and 8% perrhenate. Because these impurities could be re-
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TABLE 1
Comparative F6 – 679/125I-AG 8.1 Versus F6 – 679/188Re-AG 8.0 Biodistribution After Injection in Nude Mouse Model
Grafted Subcutaneously with Human Colon Carcinoma Cell Line (LS-174-T)
Time
5 min
%ID/g
Tumor
Blood
Liver
Kidney
Intestine
Spleen
Bone
125I-AG
8.1
4.80 6 1.21
6.49 6 1.15
2.20 6 0.50
8.12 6 3.20
1.32 6 0.37
1.30 6 0.24
1.32 6 0.31
188Re-AG
1h
8.0
2.44 6 0.93
5.02 6 0.94
8.12 6 2.36
4.24 6 0.88
3.34 6 2.55
1.04 6 0.24
1.31 6 0.27
125I-AG
8.1
6.88 6 1.09
3.58 6 0.63
0.93 6 0.03
3.11 6 0.15
1.43 6 0.67
0.62 6 0.07
0.62 6 0.13
5h
188Re-AG
8.0
4.01 6 0.41
2.59 6 0.53
0.97 6 0.30
1.29 6 0.35
16.7 6 13.4
0.43 6 0.09
0.54 6 0.17
125I-AG
8.1
10.4 6 1.6
3.03 6 0.56
0.79 6 0.09
2.06 6 0.17
0.38 6 0.09
0.55 6 0.09
0.38 6 0.05
24 h
188Re-AG
8.0
5.42 6 0.58
1.58 6 0.37
0.50 6 0.05
0.75 6 0.05
0.27 6 0.04
0.34 6 0.10
0.32 6 0.09
125I-AG
8.1
7.92 6 2.78
1.32 6 0.29
0.74 6 0.27
1.47 6 0.33
0.17 6 0.05
0.48 6 0.17
0.28 6 0.09
48 h
188Re-AG
8.0
4.04 6 1.42
0.36 6 0.21
0.25 6 0.10
0.29 6 0.08
0.12 6 0.03
0.14 6 0.02
0.23 6 0.05
125I-AG
8.1
5.92 6 0.61
0.35 6 0.12
0.46 6 0.04
1.03 6 0.17
0.07 6 0.01
0.24 6 0.02
0.09 6 0.02
188Re-AG
8.0
2.12 6 0.76
0.39 6 0.02
0.15 6 0.05
0.20 6 0.04
0.10 6 0.03
0.19 6 0.02
0.29 6 0.06
Data are expressed as mean 6 SD.
moved easily during the purification step for the radiolabeled hapten, the solution was used in this state. Under these
conditions, the uptake yield after addition of 1 GBq 188Reglucoheptonate to 21 nmol AG 8.0 was 72%. The specific
activity of 188Re-AG 8.0 after purification was estimated at
90% by chromatography. The immunoreactive fraction was
measured to 91%. The major impurities detected by chromatography could not be identified. The purification step
allowed elimination of perrhenate and 188Re hydrolysates.
Animal Study
Biodistribution Study. The results obtained (Table 1)
show that the maximum injected dose in all tissues appeared
at the same time point for both radionuclide-associated
haptens: 5 min for blood, liver, kidney, spleen, and bone;
1 h for intestine; and 5 h for tumor. The percentage of tumor
uptake of 125I-AG 8.1 was always statistically greater than
that of 188Re-AG 8.0 (P , 0.01 for all time points). The
distribution of radioactivity was comparable for blood,
spleen, and bone up to 1 h after injection and then became
statistically greater for 125I-AG 8.1 than for 188Re-AG 8.0 —
except for bone, in which uptake of 188Re-AG 8.0 was
greater than that of 125I-AG 8.1 at 48 h (P , 0.01).
The corresponding tumor-to-tissue ratios (Fig. 3) reflected these uptake values. They were higher with 125I-AG
8.1 (Fig. 3A) at 5 min for blood, spleen, and bone (P ,
0.05) and were comparable with the ratios of 188Re-AG 8.0
(Fig. 3B) for these same tissues at 1, 5, and 24 h. At late
times (48 h), 188Re-AG 8.0 showed ratios that were lower
than those for 125I-AG 8.1, except in liver and kidney (P ,
1024), which were indicative of higher tumor uptake and
lower bone uptake for 125I-AG 8.1. At 5 min, liver uptake of
188Re-AG 8.0 was higher than that of 125I-AG 8.1, resulting
in very low tumor-to-liver ratios (P , 0.01), whereas at 5,
24, and 48 h uptake was greater for 125I-AG 8.1 (P , 0.05),
with comparable tumor-to-liver ratios for both substances
(P . 0.05). In the intestine, uptake of 125I-AG 8.1 and
188Re-AG 8.0 was not significantly different at any time
point, except at 1 h when uptake was markedly greater for
188Re-AG 8.0 (P , 0.01), with a very large SD indicative of
considerable interindividual variation. In kidney, regardless
of analysis time, uptake percentages for 125I-AG 8.1 were
greater than those for 188Re-AG 8.0 (P , 0.05). Despite
high tumor uptake of 125I-AG 8.1, except at 5 min, the
tumor-to-kidney ratios obtained with 188Re-AG 8.0 were
greater than those for 125I-AG 8.1 (P , 0.05).
In summary, tumor-to-tissue ratios were highest for
188Re-AG 8.0 at 24 h, except for the tumor-to-bone ratio,
which was highest at 5 h. For 125I-AG 8.1, the tumor-toblood ratio was highest at 48 h, whereas the other ratios
were highest between 5 and 48 h.
188Re-AG 8.0 Stability. The results for immunoreactive
measurements are shown in Figure 4. Analysis of the stability of the purified 188Re-AG 8.0 solution over time
showed that the 188Re-labeled peptide, used as a control, lost
a large part of its immunoreactivity at early times. It decreased from 90.8% at 5 min to 17.7% at 5 h but remained
quite constant after that time point: 14.2% at 24 h and 8%
at 48 h.
The immunoreactive fraction in urine was around 38% at
5 min and then became very low (7.1%) between 1 and 5 h
before rising to 44% at 24 h and dropping slightly to 36% at
48 h. Most of the radioactivity was eliminated rapidly. The
immunoreactive fractions were quite stable in blood, ranging from 84% at 5 min to 26.7% at 48 h. This phenomenon
could have been caused by radiolysis resulting from the
high activity used or by instability of the radiolabeled hapten (or both).
The study of immunoreactive fractions relating to 188Relabeled hapten showed that urinary radioactivity was eliminated mainly in the form of nonimmunoreactive metabolites, whereas circulating forms in the blood were composed
mainly of immunoreactive hapten until 5 h after injection
and were then considerably reduced at late times.
Dosimetry Study. The results of the dosimetric study are
shown in Table 2. For each tissue and tumor, the cumulative
concentration is indicated in kBq/s/kg, as extrapolated by
exponential adjustment of kinetic values and the constant of
the dose corresponding to each radionuclide studied. Cal-
BISPECIFIC MONOCLONAL ANTIBODY
AND 188RE-LABELED
HAPTEN • Gestin et al.
149
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FIGURE 3. Comparison of tumor-totissue ratios obtained with 125I-AG 8.1 (A)
and 188Re-AG 8.0 (B) after injection in
nude mouse model grafted subcutaneously with human colon carcinoma cell
line (LS-174-T). Data are expressed as
mean 6 SD.
culations for 131I were performed on the basis of results for
125I, assuming that the kinetics of AG 8.1 kinetics was
identical for the two iodine radioisotopes.
With 188Re, the highest doses were obtained for tumor,
intestine, blood, and kidney, whereas with 125I the most
irradiated tissues were tumor, kidney, blood, and liver. The
least favorable tumor-to-normal tissue ratios were 8.1 and
8.5 for 131I (tumor-to-blood ratio and tumor-to-kidney ratio,
respectively) and 2.3 for 188Re (tumor-to-intestine ratio).
DISCUSSION
The use of mercaptoacetyltriglycine (MAG3) derivatives
for the radiolabeling of proteins with 99mTc or 188Re is one
of the most common techniques (28 –32). With 188Re, the
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usual approach is to radiolabel MAG3 first and then purify
the 188Re-MAG3 thus obtained before activating an acid
residue (generally butyric acid). The radiolabeling of proteins is then achieved by incubating the activated, radiolabeled MAG3 with a slightly alkaline protein solution
(28,29). Although this procedure provides radiolabeling
with high radiochemical purity, it is time-consuming and
thus unsuitable in terms of the short physical half-life of
188Re. Radiolabeling of antibodies with 188Re can be performed directly (7–9,33), but the radioantibodies obtained
generally lack stability in vivo (8,29). To avoid these drawbacks, the modified derivative of AG 8.1 used in this study
had a structure derived from MAG3 but could be radiolabeled directly with 188Re. Moreover, to facilitate the radio-
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FIGURE 4. Analysis of stability of
188Re-AG 8.0 by measurement of serum
and urinary immunoreactive fractions after injection in nude mouse model grafted
subcutaneously with human colon carcinoma cell line (LS-174-T).
labeling of this AG 8.0 peptide, a ligand-exchange technique was developed, which allowed 188Re-AG 8.0 to be
obtained in 15 min with good yields. As a reference, we
used another peptide closely related to AG 8.0, which was
labeled with a low specific activity of 125I to minimize
radiolysis (11.1 MBq/nmol). AG 8.0 was labeled with a
higher specific activity of 188Re (31.82 MBq/nmol), which
should make 188Re-AG 8.0 suitable for future RIT protocols.
The same purification technique used for labeling both
radionuclides afforded excellent results for 125I-AG 8.1 but
was less efficient in removing impurities from the 188Re-AG
8.0 solution. However, the latter was used because radiochemical purity was better than or equal to that reported for
direct radiolabeling of antibodies with 188Re (60%– 80% as
measured by the immunoreactive fraction) (7–9).
The results obtained with 125I-AG 8.1 were comparable
with those reported in the same animal model (34) for a
radiolabeled peptide that was only slightly different from
the one used in this study. The results for tumor uptake of
188Re-AG 8.0 were poorer than those with 125I labeling but
were comparable with values in the literature for antibodies
radiolabeled directly with 188Re (16,29). Kinetic analysis of
biodistribution showed that the blood clearance of 125I-AG
8.1 was lower than that of 188Re-AG 8.0 (1.23 mL/h vs. 2.1
mL/h). The higher blood clearance of AG 8.0 was apparently one of the reasons for lower tumor uptake with this
peptide than with AG 8.1. However, the radiochemical
purity of 188Re-AG 8.0 decreased very rapidly over time
when it was concentrated in the purification medium, which
suggests that impurities formed during the period required
for preparation and injection of 188Re-AG 8.0 into the animal. These impurities associated with radiolabeling complexation instability or radiolysis (or both) were apparently
responsible for part of the increase in the renal clearance of
radioactivity and for reduced tumor uptake. This was confirmed by the study of urinary immunoreactive fractions,
which showed that only 8% of the radioactivity consisted of
hapten that was recognized by antibody 679 at 1 h, whereas
this fraction was .30% at all other time points. This difference may be attributed to rapid urinary elimination of
TABLE 2
Dosimetry Estimations Obtained from Biodistribution Results for 125I-AG 8.1 and 188Re-AG 8.0
for Theoretic Injection of 3.7 MBq 131I or 188Re in Nude Mouse Model Grafted Subcutaneously
with Human Colon Carcinoma Cell Line (LS-174-T)
131I
188Re
Tissue
Ccumulative
(kBq/s/kg)
D (cGy)
Tumor/nontumor
dosimetry ratio
Ccumulative
(kBq/s/kg)
D (cGy)
Tumor/nontumor
dosimetry ratio
Tumor
Blood
Liver
Kidney
Intestine
Spleen
Bone
6.59
0.82
0.41
0.78
0.13
0.26
0.13
259.79
32.16
16.06
30.65
5.21
10.37
5.29
8.1
16.2
8.5
49.9
25
49.1
1.07
0.28
0.14
0.15
0.47
0.06
0.07
172.63
45.39
22.76
25.04
75.34
10.42
12.24
3.8
7.6
6.9
2.3
16.6
14.1
BISPECIFIC MONOCLONAL ANTIBODY
AND 188RE-LABELED
HAPTEN • Gestin et al.
151
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impurities caused by the release of noncomplexed 188Re
from 188Re-AG 8.0 before administration. At later time
points, the metabolism of this radiolabeled peptide was a
contributing factor, although a highly significant fraction of
peptide circulating in the blood remained immunoreactive
over time. Moreover, the results of the biodistribution study
show that 188Re-AG 8.0 had very high hepatic and intestinal
uptake at early time points compared with that of 125I-AG
8.1, apparently because of biliary elimination of 188Re-AG
8.0 radioactivity. This mode of elimination could have
caused increased clearance of the radiolabeled peptide and
thus decreased tumor uptake at later times. It appears that
125I-AG 8.1 was eliminated preferentially by the renal route.
In fact, the percentages of renal uptake were always greater
for 125I-AG 8.1 than for 188Re-AG 8.0 at all time points.
Finally, bone uptake was greater at 48 h with 188Re-AG 8.0
than with 125I-AG 8.1, possibly because 188Re-AG 8.0
lacked labeling stability in vivo. This could have caused
local uptake by exchange between the 188Re-MAG3 complex of the peptide and the phosphocalcic structures of the
bone network.
For all normal tissues studied, the %ID/g with 125I-AG
8.1 was lower than that for antibody radiolabeled directly
with 125I (5,16,35,36). This phenomenon, which has been
reported for other bispecific antibody or hapten pairs
(14,15), resulted in tumor-to-tissue ratios that were greater
than those observed with antibodies that were radiolabeled
directly (16,28,37). Comparison of the biodistribution obtained with AG 8.1 labeled with 125I and 188Re showed
differences in the in vivo distribution of radioactivity depending on the radioelement used. In fact, this phenomenon
could have been attributed to the structural difference between the two haptens. The elimination kinetics of 188ReAG 8.0 appeared to be faster than that of 125I-AG 8.1, partly
because 188Re-AG 8.0 has significant hepatobiliary clearance.
Two assumptions were made in this study relative to
dosimetry calculations. First, the effect of g emissions was
disregarded at the doses delivered because of the low proportion of photon emissions by 188Re (9.18 3 10215 kg 3
Gy/Bq 3 s) compared with particle emissions (1.24 3 10213
kg 3 Gy/Bq 3 s) and the fact that this energy was deposited
at greater distances than with b2 emissions. Second, particle
emissions were considered nonpenetrating in the geometry
of our experimental conditions. This assumption, though
frequently made for dosimetry in vivo in humans, cannot be
verified in mice. Thus, our results need to be interpreted
with caution, given the size of murine organs and tissues,
which are generally smaller than the maximal range of b2
particles of 188Re (6). Nevertheless, it is necessary to make
these calculations to compare the results obtained with both
radionuclides with those published using the same animal
model (21–24).
On the whole, our results are encouraging for the development of RIT protocols with this technique, even if improvement of 188Re-AG 8.0 stability is still necessary. Un-
152
THE JOURNAL
OF
der these conditions, the injection of quantities of
radioactivity three to four times higher than the quantities
we used may allow the delivery of doses approximating
those reported in other studies relating to this method of
human tumor treatment (33,38,39).
CONCLUSION
This study indicates that 188Re can be used for the radiolabeling of hapten in two-step RIT protocols with the AES.
The main advantage of this radionuclide over 131I is its
range, which should allow the treatment of solid tumors
around 1 cm in diameter. However, the method used for
hapten radiolabeling did not provide optimal tumor uptake.
Other rhenium-chelating agents easily substituted for the
N-terminal end of AG 8.1 could offer improved labeling
efficiency and in vivo stability.
ACKNOWLEDGMENTS
The authors thank James Gray for technical assistance.
This study was supported by a grant from the Comité
Départemental de la Ligue Nationale Contre le Cancer,
Département de la Vendée, and by a grant from the Comité
Départemental de la Ligue Nationale Contre le Cancer,
Département des Deux-Sèvres.
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BISPECIFIC MONOCLONAL ANTIBODY
AND 188RE-LABELED
HAPTEN • Gestin et al.
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Two-Step Targeting of Xenografted Colon Carcinoma Using a Bispecific Antibody
and 188Re-Labeled Bivalent Hapten: Biodistribution and Dosimetry Studies
Jean F. Gestin, Anthony Loussouarn, Manuel Bardiès, Emmanuel Gautherot, Anne Gruaz-Guyon, Catherine
Saï-Maurel, Jacques Barbet, Chantal Curtet, Jean F. Chatal and Alain Faivre-Chauvet
J Nucl Med. 2001;42:146-153.
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