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Experimental Therapeutics |
Departments of Radiology [C. F. F., C. J. R., M. R. Z.] and Pathology [D. D. B., M. R. Z.], Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EGFR cell line indicated that internalized and
total cell-associated activity for the 125I-labeled
D-KRYRR-L8A4 conjugate were up to 4 and 5 times higher,
respectively, than for L8A4 labeled with 131I using
Iodogen. Paired-label comparisons in athymic mice with s.c. U87EGFR
xenografts demonstrated up to 5-fold higher tumor uptake for mAb
labeled using D-KRYRR. Higher levels of radioiodine
activity also were observed in kidney when L8A4 was labeled using
D-KRYRR. Another paired-label study directly compared L8A4
labeled using radioiodinated D-KRYRR and
L-KRYRR, and confirmed the role of D-amino
acids in enhancing tumor uptake. These results suggest that
D-KRYRR is a promising reagent for the radioiodination of
internalizing mAbs, such as the anti-EGFRvIII mAb L8A4. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to overexpression, oncogenic transformation can lead to rearrangement of the EGFR gene, potentially yielding molecular targets with greater tumor specificity than the wild-type EGFR (2) . Of particular interest for targeting applications is EGFRvIII, which is characterized by a genomic deletion of amino acid residues 6–273 from the extracellular domain of the EGFR with the formation of a novel glycine at the new fusion junction (3 , 4) . This 145-kDa truncated receptor has been identified on gliomas and breast, lung, and ovarian carcinomas but not on normal human tissues (5 , 6) . Importantly, in 4 of 5 human glioma biopsies, expression of EGFRvIII was 270,000–680,000 receptors per cell, a level that should be sufficient for effective targeting (7) .
We have developed a series of murine mAbs including L8A4 that bind selectively to EGFRvIII-expressing cells and tumor xenografts but not to normal tissue (6) . In vitro and in vivo studies have shown that these anti-EGFRvIII mAbs are rapidly internalized after binding to cell surface receptor, resulting in degradation of the mAb inside the tumor cell (8) . For this reason, the radioiodination method used to label anti-EGFRvIII mAbs could have a major impact on their potential utility for radioimmunotherapy.
Labeling the anti-EGFRvIII mAbs L8A4 and H10 by a direct iodination method (Iodogen) resulted in only modest tumor:normal tissue ratios in athymic mice bearing EGFRvIII-expressing HC2 20 d2 xenografts (8) . Coupling radiolabeled, inert disaccharides, such as tyramine-cellobiose, to mAbs has been reported to increase the retention of radioiodine in tumor cells after intracellular mAb processing (9) . With mAbs L8A4 and H10, this approach improved tumor activity levels compared with Iodogen labeling; however, low tumor localization indices were observed (8) .
We have been investigating an alternative approach for labeling internalizing mAbs that involves the conjugation of a labeled prosthetic group to the protein that is positively charged at lysosomal pH. Because positively charged molecules are often used as lysosomal markers (10) , we hypothesized that positively charged catabolites generated during mAb proteolysis should be retained in lysosomes. The feasibility of this strategy was demonstrated in a series of experiments in which the anti-EGFRvIII mAb L8A4 was radioiodinated using SIPC (11 , 12) . HPLC analyses of cell lysates demonstrated that radioiodine activity was retained in these EGFRvIII-expressing cells in the form of iodonicotinic acid-lysine, a positively charged species. Although SIPC labeling enhanced the retention of radioiodine activity in EGFRvIII-expressing cells in vitro and yielded higher activity levels in tumor xenografts compared with mAb labeled using Iodogen, these advantages were of short duration.
The current study was performed to determine whether coupling the mAb to a labeled peptide prosthetic group containing multiple positively charged amino acids would result in prolonged enhancement of tumor uptake. Prototypic pentapeptides with the sequence Lys-Arg-Tyr-Arg-Arg (KRYRR) composed entirely of both D- and L-amino acids were evaluated.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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General Chemical Methods.
Analytical HPLC analyses were conducted on a Beckman system
(model 126 pump, model 168 UV detector, and model 170 radioactivity
detector) connected to a model 406 analogue interface module. The
chromatographic system consisted of a reverse-phase C18-Basic column (5
µm, 4.6 x 250 mm), obtained from YMC (Wilmington,
NC), eluted in gradient mode at a flow rate of 1 ml/min. The gradient
used 0.1% trifluoroacetic acid in water as solvent A and 0.1%
trifluoroacetic acid in acetonitrile as solvent B. Starting with 100%
A for 1 min, the gradient was decreased to 35% A over 25 min, held for
5 min, changed to 100% B for 15 min, and finally reequilibrated at
100% A for 30 min. Radioactivity was quantified using a LKB 1282
Compugamma (Wallac, Turku, Finland) dual-channel, automated gamma
counter. The higher levels of radioactivity used in the radiosyntheses
were measured with a Capintec-7R radioisotope calibrator.
mAbs and Cell Lines.
The characterization and purification of murine L8A4, an IgG1 that
binds specifically to EGFRvIII with an affinity of 2 x 109 M–1, have
been detailed in previous publications (6
, 13) . The
EGFRvIII-expressing U87 MGEGFR cell line has been described by
Nishikawa et al. (14)
and was generated by
transfecting the U87 MG human glioma cell line with a vector containing
the cDNA for EGFRvIII. Cells were grown in zinc option media
(Life Technologies, Inc., Grand Island, NY), containing 10%
fetal bovine serum and 600 µg/ml Geneticin sulfate. U87
MGEGFR cells express approximately (4–13 x 105 EGFRvIII receptors per cell in culture
(12)
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Radioiodination of KRYRR Peptides.
Na[125I]iodide or
Na[131I]iodide (1 mCi) was added to
D- or L-KRYRR (10 µl, 1 mg/ml PBS) diluted
with PBS (60 µl) in a glass vial coated with Iodogen (100 µg). The
vial was placed on an orbital shaker for 40 min, and then the mixture
was diluted with 3 ml of water. The solution was eluted through a tC18
cartridge, which was rinsed with an extra 3 ml of water. The
radioiodinated peptide was eluted in 400 µl of acetonitrile
containing 1% acetic acid. The solvent was evaporated under a stream
of nitrogen prior to reaction with the mAb.
Conjugation of Radioiodinated Peptide to mAb.
As shown in Fig. 1
, peptide activation was achieved by addition of s-SMCC (25 µl, 2
mg/ml PBS), and 0.1 M borate buffer (200 µl) to the vial
containing the labeled peptide and reacting for 30 min at room
temperature. Meanwhile, to add free sulfhydryl groups to L8A4, the mAb
(69 µl, 2.88 mg/ml PBS) and 2-iminothiolane (6 µl, 3 mg/ml) were
incubated in PBS (100 µl) for 30 min at room temperature. The
thiolated mAb was purified using a 3-ml Sephadex G-25 spin column.
Thiolated mAb was added directly to the vial containing the
maleimido-activated, radioiodinated KRYRR, and the coupling was allowed
to proceed at room temperature for 45 min, at which point the reaction
was quenched by the addition of an excess of iodoacetamide (10 µl,
100 mg/ml PBS). After 20 min, the mixture was purified over a PD-10
column eluted with PBS.
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Evaluation of Immunoreactivity.
Streptavidin-coated magnetic beads (1 ml) were mixed with the
biotinylated recombinant extracellular domain of EGFRvIII (100–150
µl, 1 mg/ml) for 3 h at room temperature and rinsed with 115
mM phosphate buffer, pH 7.4, containing 0.05% Brij 35 and
0.05% BSA to remove unbound proteins. The beads were resuspended in 1
ml of this buffer and stored at 4°C. Control beads were fabricated as
described above except that biotinylated BSA was conjugated to the
beads. The immunoreactive fraction was determined by incubating the
labeled L8A4 mAb preparations (approximately 20,000 cpm in 20 µl), in
triplicate with increasing volumes of beads (10, 20, and 40 µl) in
500 µl of 115 mM PBS. After a 45-min agitation at room
temperature, the beads were isolated from the supernatant using a
magnetic separator. Beads and supernatants were counted for
radioactivity, and specific binding was determined by subtracting
nonspecific binding to BSA beads from the binding to EGFRvIII beads.
The immunoreactive fraction was calculated according the method of
Lindmo et al. (15)
by plotting the inverse of
the percentage of specific binding versus the inverse of the
bead volume.
Assay for mAb Internalization in Vitro.
The internalization and processing of
125I-labeled D-KRYRR-L8A4 and L8A4
labeled with 131I using the Iodogen method were
compared in a paired-label experiment as described in previous
publications (8
, 13)
. Approximately 1 µg of each mAb was
incubated with 1 x 106 U87
MGEGFR cells for 1 h at 4°C and then washed with cold zinc
option culture media containing 10% FCS. The cells were then
resuspended in media/10% FCS to a density of 3 x 106 cells/ml, and the culture temperature was
raised to 37°C. Aliquots of cells in triplicate were removed at 2, 6,
and 24 h and centrifuged, and the supernatants were saved for
counting. The cell pellet was washed twice at 4°C for 15 min with
zinc option culture media containing 10% FCS (adjusted to pH 2.0 with
HCl) to remove cell surface-associated activity. The acid washes were
combined and counted along with the culture supernatants and cell
pellets in the dual-channel gamma counter. The supernatants were
incubated with 12.5% trichloroacetic acid to determine
protein-associated activity. The counts in each compartment were
expressed as a percentage of the activity bound to the cells
immediately after the 1-h incubation at 4°C.
Tissue Distribution of Radioiodinated KRYRR Peptides.
The biodistribution of the labeled peptides was investigated in BALB/C
mice weighing 20–25 g. In the first experiment, groups of five animals
were injected i.v. with 5 µCi of 125I-labeled
L-KRYRR and 5 µCi of 131I-labeled
D-KRYRR and killed by halothane overdose 1, 2, 4, and
24 h later. A second study was performed to determine the effect
of co-administration of D-lysine on radioiodine
distribution. Groups of five mice received 5 µCi of
125I-labeled D-KRYRR and 7 µCi of
131I-labeled L-KRYRR i.v. and
simultaneously were given 0, 30, or 60 mg of D-lysine via
the i.p. route. All groups were killed 2 h after injection by
halothane overdose. Organs were harvested, weighed, and counted in the
dual-channel gamma counter. The % ID/g was calculated by comparison to
injection standards of appropriate count rate.
HPLC Analysis of Peptide Catabolites in Urine.
Urine samples obtained during the first experiment were pooled at each
time point, diluted with 500 µl of PBS, and passed through a 45 µm
acetate membrane filter. The samples were analyzed on the C18-Basic
HPLC column. Fractions were collected every 15 s using a fraction
collector and assayed for 125I and
131I activity in the dual-channel gamma counter.
The fraction eluting at 15 min was further analyzed by ITLC. The plate
was eluted in saline:ethanol (95:5), cut in half, and counted for
radioactivity.
Tissue Distribution of Labeled L8A4 mAb.
Xenografts of the U87 MGEGFR cell line were established in athymic
mice by s.c. injection of 50 µl of tumor homogenate in the flank of
each mouse. Previous flow cytometric studies have shown that
disaggregated U87 MGEGFR xenografts have a mean of 2.5 x 105 EGFRvIII receptors per cell
(12)
. Mice weighing 22–26 g were injected with 1 µCi of
125I-labeled D-KRYRR-L8A4 and 2 µCi
of 131I-labeled L8A4 prepared using the Iodogen
method. Groups of five animals were killed by halothane overdose 12,
24, 36, 48, and 72 h later. In the second experiment, mice
weighing 14–22 g were injected with 1.5 µCi (6 µg) each of
125I-labeled L-KRYRR-L8A4 and
131I-labeled D-KRYRR-L8A4, and groups
of five animals were necropsied at 6 and 12 h and 1, 2, 3, 5, and
6 days.
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RESULTS |
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Cell Retention and Internalization in Vitro.
The internalization and cellular processing of
125I-labeled D-KRYRR-L8A4 after
incubation at 37°C with EGFRvIII-expressing U87 MGEGFR cells was
compared with that of L8A4 labeled with 131I
using Iodogen. As shown on Fig. 2
, internalized and total cell-associated (internalized + cell
surface) radioiodine activity levels were significantly higher for
125I-labeled D-KRYRR-L8A4 at all time
points, with the differences between the two labeling methods
increasing with time. At the 2-h time point, differences in cellular
retention corresponded to differences in the release of intact mAb from
the cells. After a 24-h incubation at 37°C, the total cell-associated
activity was 5.3 times higher for
125I-labeled D-KRYRR-L8A4 at this
time (Iodogen, 11.1 ± 1.0%; D-KRYRR,
58.3 ± 12.4%). Likewise, the percentage of activity
retained as internalized counts was nearly 4 times higher for the
D-KRYRR-L8A4 conjugate (Iodogen, 5.0 ± 1.2%; D-KRYRR, 18.8 ± 5.4%). With
125I-labeled D-KRYRR-L8A4, there was
a corresponding decrease in the fraction of mAb initially bound to U87
MGEGFR cells released into the cell culture supernatant as labeled
degradation products (trichloroacetic acid-soluble counts) into the
cell culture supernatant. For example, degraded counts in the
supernatant at 24 h accounted for 19.7 ± 6.8 and
51.1 ± 6.3% of total counts for L8A4 labeled via
D-KRYRR and Iodogen, respectively.
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). With the exception of the kidneys, radioactivity levels generally
were significantly lower for the D-amino acid
peptide. Approximately 30% ID of 131I-labeled
D-KRYRR was retained in the kidneys during the
first 4 h after injection, declining to about half this level by
24 h. In contrast, kidney uptake of
125I-labeled L-KRYRR was
1.30 ± 0.27% ID at 1 h and declined rapidly
thereafter. On the other hand, retention of
131I-labeled D-KRYRR in
lungs, blood, thyroid, and stomach, as well as intestines, muscle,
heart, and brain (data not shown), was significantly lower
(P < 0.05, paired t test) than
retention of 125I-labeled L-KRYRR from 1
to 4 h, and in some cases, at 24 h after injection. Because
of the proclivity of free iodide for the thyroid, accumulation of
radioiodine activity in the thyroid can reflect dehalogenation. The %
ID of 131I in the thyroid 0.2% ID or less at all
time points, levels significantly lower than those observed for
125I (2.65 ± 0.50 to
5.24 ± 1.26%).
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). Liver activity levels were somewhat higher in animals receiving
D-lysine, but these differences were not statistically
significant.
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5 min. As shown in Fig. 5
, there was little evidence for free iodide in the urine from the
D-KRYRR (0.8% at 1 h to 4.2% at 24 h), whereas
free iodide generated from L-KRYRR ranged from 25 to 69%.
Because both iodotyrosine and the radioiodinated KRYRR peptide eluted
at 13 min (Fig. 4
), ITLC was used to differentiate between these
species. ITLC analysis revealed that 18–22% of the labeled
L-KRYRR was excreted as iodotyrosine, and 82–98% of the
urine activity from 131I-labeled
D-KRYRR was present as intact peptide.
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EGFR xenografts to determine the effect of
labeling method on tumor and normal tissue accumulation of radioiodine
activity. Direct comparison of 125I-labeled
D-KRYRR-L8A4 and L8A4 labeled with
131I using Iodogen demonstrated that use of the
D-amino acid prosthetic group resulted in significantly
higher (P < 0.05) tumor levels at all time
points (Fig. 6
). The peptide labeling method increased the tumor retention of
radioiodine activity by factors of approximately 2 at 12 h, 3 at
24 h, and 5 at 36 h, 48 h, and 72 h. Maximum tumor
accumulation occurred at 24 h for
125I-labeled D-KRYRR-L8A4
(21.5 ± 3.9% ID/g) and at 12 h for L8A4 labeled
with 131I using Iodogen (7.99 ± 0.84% ID/g). The % ID/g in tumor for the peptide-mAb conjugate
declined about 2-fold from 24 to 72 h, presumably reflecting the
rapid growth of this xenograft (tumor weight at necropsy: 24 h,
1.06 ± 0.24 g; 72 h, 2.21 ± 0.64 g).
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. With 125I-labeled D-KRYRR-L8A4,
liver, spleen, and kidney levels generally were significantly higher
than those for mAb labeled using Iodogen, whereas thyroid, blood,
stomach, muscle, and heart levels were significantly lower. Thyroid
levels for 125I-labeled D-KRYRR-L8A4
were 52 ± 9, 29 ± 5, 17 ± 1, 11 ± 1, and 11 ± 2% of the
levels for mAb labeled using Iodogen at 12, 24, 36, 48, and 72 h,
respectively. Tumor:blood ratios for D-KRYRR-L8A4 increased
from 1.8:1 at 12 h to 16.7:1 at 72 h, compared with 0.6:1 and
1.8:1, respectively, for mAb labeled using Iodogen.
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. Compared with L-KRYRR, use of radioiodinated
D-KRYRR increased the tumor retention of radioactivity by
301 ± 14, 418 ± 167, 657 ± 80, 956 ± 222, and 1525 ± 663%
on days 1, 2, 3, 5, and 6, respectively. Maximum tumor accumulation for
both conjugates was observed at 24 h (D-KRYRR,
55.6 ± 8.7% ID/g; L-KRYRR, 18.4 ± 2.5% ID/g) and declined by a factor of about 2.7 for the
D-KRYRR-L8A4 conjugate by day 6 (Table 2)
. Again, rapid
xenograft growth (day 1, 0.12 ± 0.04 g; day 6,
1.58 ± 1.07 g) probably contributed to the decline
in % ID/g radioiodine activity in tumor with time. Normal tissue
levels for the D-KRYRR-L8A4 conjugate generally were
significantly higher than for the L-KRYRR-L8A4 conjugate
except in the stomach and the thyroid. Uptake of radioiodine in the
thyroid ranged from 0.24 ± 0.01% ID to 0.37 ± 0.10% ID for D-KRYRR-L8A4 compared with a range of
0.70 ± 0.12 to 3.56 ± 0.46% ID for
L-KRYRR-L8A4 conjugate. Tumor:normal tissue ratios for
131I-labeled D-KRYRR-L8A4 and
125I-labeled L-KRYRR-L8A4 are
presented in Fig. 7
. Except for the kidney at later time points, significantly higher
tumor:normal tissue ratios were observed for the D-peptide
conjugate. Optimal selectivity of radioiodine uptake generally occurred
2–3 days after injection, before tumors had reached 1 g in
weight.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Herein, we have investigated whether radioiodination of L8A4 using a D-amino acid peptide containing several positively charged amino acids would improve the retention of radioiodine activity in EGFRvIII-expressing tumor cells. Lysosomal proteases readily degrade proteins composed of L-amino acids but D-amino acids are not substrates for endogenous proteases (18 , 19) . The prototypical peptide selected for investigation was D-KRYRR. A tyrosine was included for facile radioiodination, and a lysine was added at the N-terminal to permit convenient coupling of the peptide to the mAb. To add three positive charges to the peptide, three arginine residues were chosen to add positive charges because the arginine side chain is the most basic (pKa = 13.2) of the naturally occurring amino acids.
Our rationale for using a polycationic peptide is based on several observations. First, chloroquine and other dyes with multiple positive charges are retained in lysosomes to such a degree that they are used to stain this organelle (10) . Second, when a series of D-dipeptides were introduced into lysosomes by endocytosis, a positively charged dipeptide was trapped, whereas six neutral dipeptides were not (19) . Finally, radioiodine was retained in tumor cells as positively charged, lower molecular weight molecules when mAb L8A4 was labeled using SIPC (11) .
The in vivo behavior of radioiodinated D-KRYRR and L-KRYRR were compared in normal mice to facilitate interpretation of KRYRR-mAb conjugate biodistributions. Moreover, only limited information is available concerning the tissue distribution of D-amino acid peptides. Pappenheimer et al. (20 , 21) evaluated the excretion of five octapeptides composed of D-amino acids of varying charge and lipophilicity. Peptides containing serine or leucine exhibited slower whole body clearances compared to more hydrophilic peptides, suggesting that exclusion of lipophilic amino acids should be a design criteria for labeled D-amino acid peptide acylation agents.
A potential concern with labeling peptides by direct iodination is that previous studies have shown that L-amino acid peptides labeled in this manner are rapidly dehalogenated in vivo, as reflected by high radioiodine levels in the thyroid and stomach (22 , 23) . Our results revealed high radioiodine levels in the thyroid and stomach after injection of 125I-labeled L-KRYRR, reaching maximum values of 5.24 ± 1.26 and 8.51 ± 1.45%, respectively. The blood and normal organ distribution of 125I activity were in excellent agreement with those reported for free iodide (24) , suggesting that rapid dehalogenation of 125I-labeled L-KRYRR (or its intermediate catabolite) had taken place. Thyroid and stomach levels from co-administered 131I-labeled D-KRYRR were 1–2 orders of magnitude lower, suggesting low recognition of its D-iodotyrosine residue by endogenous deiodinases. This is in agreement with previous studies demonstrating that the deiodination of D-tyrosine was considerably lower than that of its L-enantiomer (25 , 26) .
The in vivo results also are consistent with differential sensitivity of L- and D-KRYRR to proteolysis. Chromatographic analysis of urine from mice injected with the two peptides suggests that 131I-labeled D-KRYRR was largely excreted intact, whereas radioiodinated L-KRYRR was eliminated as iodide, iodotyrosine, and other unidentified catabolites that probably correspond to proteolytically degraded peptide fragments. These results are in agreement with those reported for the renal excretion of other D-amino acid peptides (20 , 21) .
Normal tissue clearance of both peptides generally was rapid except for 131I-labeled D-KRYRR in the kidneys. The kidney plays an active role in the metabolism of peptides. These molecules can be reabsorbed by the proximal tubular cells, internalized via endocytosis, and routed to renal lysosomes. Positively charged molecules, such as D-KRYRR, are much more likely to undergo tubular absorption because the kidney luminal membrane is anionic (27) . High concentrations of cationic amino acids can inhibit this process by competing for the anionic sites on the luminal cell surfaces (28) . Indeed, basic amino acids have been used to reduce kidney uptake of radioactivity after the injection of radiolabeled mAb fragments and peptides (29 , 30) . To determine whether this approach could lower 131I-labeled D-KRYRR kidney uptake, co-administration of D-lysine was investigated. One injection of 60 mg D-lysine yielded a 3-fold reduction in kidney levels, an effect similar to that reported for a single dose of D-lysine on the renal uptake of labeled octreotide analogues (29) . Further reductions in kidney levels have been achieved through the use of optimized D-lysine administration protocols (30) .
An important objective in developing labeling strategies for L8A4 and
other internalizing mAbs is to maximize radionuclide retention in the
tumor cell after cellular processing of the mAb. For this reason, the
internalization and cellular processing of
125I-labeled D-KRYRR-L8A4 by
EGFRvIII-expressing U87EGFR cells was compared to L8A4 labeled with
125I using Iodo-Gen. Both internalized and total
cell-associated counts were significantly higher for the
D-KRYRR-mAb conjugate at all time points. After 24 h,
the percentage of activity retained as intracellular counts was nearly
4 times higher, and total cell-associated activity was 5.3 times higher
for 125I-labeled D-KRYRR-L8A4. These
results are superior to those obtained using other residualizing
methods for labeling L8A4. When the same assay was used to compare the
cellular retention of L8A4 labeled using tyramine-cellobiose and
Iodogen on the EGFRvIII-expressing HC2 20 d2 line, the maximum
advantage seen for TCB was a factor of 2.8 at 20 h
(8)
. Using the same cell line and mAb, the maximum
advantage seen for SIPC compared with Iodo-Gen was only a factor of 1.6
at 2 h, and the advantage declined rapidly thereafter
(11)
. We speculate that the excellent cellular retention
observed with the D-KRYRR-L8A4 conjugate reflects the
proteolytic generation of 125I-labeled
D-KRYRR, which, having multiple positive charges, exhibits
restricted passage through lysosome and cell membranes. Detailed
studies of the labeled catabolites generated from mAbs labeled using
D-KRYRR will be done to determine the validity of this
assumption.
The cellular processing of mAbs labeled using another acylation agent containing D-amino acids has been described recently (31) . A D-amino acid peptide was used to couple DTPA to the mAb with the goal of exploiting the fact that mAb-DTPA-radiometal conjugates frequently exhibit effective retention of the radiometal in tumor cells. When D-Gly-Tyr-Lys-DTPA was radioiodinated and coupled to internalizing mAbs, retention of radioiodine activity by tumor cells was up to 3 times higher than for the same mAb labeled using chloramine-T. The magnitude of these gains was lower than that of the gains seen with D-KRYRR; however, comparing results obtained with mAbs binding to different internalizing antigens must be done with care because of potential differences in variables such as dynamics of mAb internalization and intracellular processing. With that caution in mind, it should be noted that similar results to those described herein were obtained by labeling internalizing mAbs using dilactitol-tyramine (32) . Unfortunately, poor mAb coupling yields (3–6%) detract from the utility of this method.
Tissue distribution studies were performed in athymic mice to determine whether the increased retention of radioiodine observed in EGFRvIII-expressing tumor cells with D-KRYRR-L8A4 in vitro provided a similar advantage in vivo. Consistent with the results of the cellular processing and internalization assays, the tumor delivery enhancement achieved with D-KRYRR (relative to Iodogen labeling) was more than twice that obtained with either TCB or SIPC. When L8A4 was labeled using TCB, the tumor delivery advantage ranged between 2 and 3 between 1 and 7 days after injection (8) , whereas SIPC yielded a maximum enhancement of 1.28 at 1 day, with no improvement in tumor levels seen after 48 h (11) . In contrast, D-KRYRR imparted a 3-fold advantage at 1 day, increasing to a 5.5-fold enhancement 3 days after injection of L8A4.
In summary, the practical objective of this study was to increase the retention of radioiodine in tumor after injection of radioiodinated anti-EGFRvIII mAbs, and this was achieved. The mechanisms responsible for this effect will be confirmed in subsequent experiments. The tumor uptake of radioiodinated D-KRYRR-L8A4 was significantly higher than that of co-administered L-KRYRR-L8A4, suggesting that the inertness to proteolytic degradation of radioiodinated D-KRYRR played a critical role. To optimize this general strategy, we are investigating the effect of amino acid charge, lipophilicity, and peptide length on the in vitro and in vivo behavior of radioiodinated D-amino acid-internalizing mAb conjugates.
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FOOTNOTES |
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1 Supported in part by NIH Grants CA42324,
CA11898, and NS20023. 
2 To whom requests for reprints should be
addressed, at Department of Radiology, Box 3808, Duke University
Medical Center, Durham, NC 27710. Phone: (919) 684-7708; Fax:
(919) 684-7121.
3 The abbreviations used are: EGFR, epidermal
growth factor; EGFRvIII, EGFR variant III; mAb, monoclonal antibody;
SIPC, N-succinimidyl
5-[125/131I]iodo-3-pyridinecarboxylate; s-SMCC,
sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate; BSA,
bovine serum albumin; HPLC, high-performance liquid chromatography;
ITLC, instant thin layer chromatography; % ID/g, percentage of the
injected dose/g of tissue; DTPA, diethylenetriaminepentaacetic acid.
Received 10/25/99. Accepted 6/12/00.
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 F. G. van Schaijk, M. Broekema, E. Oosterwijk, J. E.M. van Eerd, B. J. McBride, D. M. Goldenberg, F. H.M. Corstens, and O. C. Boerman Residualizing Iodine Markedly Improved Tumor Targeting Using Bispecific Antibody-Based Pretargeting J. Nucl. Med., June 1, 2005; 46(6): 1016 - 1022. [Abstract] [Full Text] [PDF]
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R. Stein, S. V. Govindan, M. Hayes, G. L. Griffiths, H. J. Hansen, I. D. Horak, and D. M. Goldenberg Advantage of a Residualizing Iodine Radiolabel in the Therapy of a Colon Cancer Xenograft Targeted with an Anticarcinoembryonic Antigen Monoclonal Antibody Clin. Cancer Res., April 1, 2005; 11(7): 2727 - 2734. [Abstract] [Full Text] [PDF]
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S. V. Govindan, G. L. Griffiths, R. Stein, P. Andrews, R. M. Sharkey, H. J. Hansen, I. D. Horak, and D. M. Goldenberg Clinical-Scale Radiolabeling of a Humanized Anticarcinoembryonic Antigen Monoclonal Antibody, hMN-14, with Residualizing 131I for Use in Radioimmunotherapy J. Nucl. Med., January 1, 2005; 46(1): 153 - 159. [Abstract] [Full Text] [PDF]
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 Z. Cheng, J. Chen, T. P. Quinn, and S. S. Jurisson Radioiodination of Rhenium Cyclized {alpha}-Melanocyte-Stimulating Hormone Resulting in Enhanced Radioactivity Localization and Retention in Melanoma Cancer Res., February 15, 2004; 64(4): 1411 - 1418. [Abstract] [Full Text] [PDF]
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) and
L8A4 labeled with 131I using Iodo-Gen (
). Shown
are the percentages of the radioiodine counts that are cell
associated, internalized, and released as degraded or intact mAb into
the cell culture supernatant. Columns, average of
triplicates; bars, SD.
) in normal mice.
Data points, average of five mice per time point;
bars, SD.
) in normal mice.
Data points, average of five mice per time point;
bars, SD.
