(CANCERRESEARCH
55,5288—5295,
November15, 19951
Lysine Reduces Renal Accumulation of Radioactivity Associated with Injection of
the [‘77Lu]a-[2-(4-Aminophenyl)ethyl]-1,4,7,1O-tetraaza-cyclodecane-1,4,7,1Otetraacetic acid-CC49 Fab Radioimmunoconjugate
Louis R. DePalatis,'
Kevin A Frazier,
Roberta
C. Cheng, and Nicolas J Kotite2
Bioproducts and Materials Research and Development Laboratories, Dow Chemical Co., Midland, Michigan 48674
ABSTRACT
The high uptake and prolonged renal retention of monoclonal antibody
fragments that are conjugated with radlometal chelates precludes their
routine clinical use due to high background
counts, which may hinder
detection of nearby lesions and/or cause renal radiotoxicity. We report on
the potential use of Lys as a pharmacological
agent to enhance renal
excretion
of the
[‘“LuJa-[2-(4-aminophenyl)
ethyl]-1,4,7,1O-tetraaza-cy
clodecane-1,4,7,1O-tetraacetlc acid CC49 Fab ([‘@LuJCC49
Fab) radloim
munoconjugate. The monoclonal antibody portion of this complex is
directed toward the tumor-assodated glycoproteln-72 antigen. Lys was
administered to female BALB/c mice by i.p. InjectIons. [@Lu]CC49 Fab
bolus injections were given by the i.v. route. ResUltSof our investigations
showed that: (a) kidney radioactivity concentrations
were inversely re
lated to Lys dose. The optimal dose (50 mg/mouse) evoked a 3-fold
reduction in kidney counts; (b) Lys was most effective when injected 15
90Y,
‘86Re, ‘53Sm, and
‘77Lu are very
stable
in vivo because
of their
was both rapid (3-folddecrease at 15 mm after injection)and prolonged
high affinity constants for their respective chelating agents and the
stable chemical bonds formed between functionalized groups on the
chelating agents and amino acid side chains of the antibody molecule
(4-fold decrease at 24 h after injection); (d) a single Lys dose decreased
total body radioactivity by >2.5-fold; (e) urine excretion of radioactivity
chelating agents are not metabolized and cleared from tissues to the
was enhanced
in Lys-treated
same degree as commonly used radionuclides of the halogen series,
analyses
a GF-250
mm before, or at the same time as, [1@LuJCC49Fab; (c) the renal effect
using
radioactivity
coeluted
mice. High pressure
column
showed
liquid chromatographic
that a large
fraction
with a [‘“LuJCC49Fab Injection
of this urine
standard.
We
conclude that Lys enhances the urinary excretion of radioactivity associ
ated with [1@Lu]CC49
Fab. These observations
warrant
further
study
with regard to the use of amino acids or their derivatives as pharmaco
logical agents to enhance
the urinary
excretion
of small-molecule
radio
immunoconjugates.
INTRODUCTION
Radiolabeled MoAbs3 have been studied extensively for their po
tential as tumor diagnostic or therapeutic agents. During the early- to
mid-1980s, in vivo studies focused on the pharmacokinetic and bio
distribution profiles of the IgG form (M1 150,000) in animal tumor
models and cancer patients. Subsequent studies compared these data
with those obtained using the enzymatically derived F(ab')2 (Mr
100,000) and Fab (Mr 50,000) forms, and the recombinant sFv form
(Mr 27,000).
Because
of their smaller
molecular
size, antibody
frag
ments exhibit higher blood clearance rates than the larger IgG parent
molecules. Thus, sFv forms have been reported to have the highest
rate of clearance when compared with those of Fab or Fab', F(ab')2,
and !gG, which show progressively slower clearance rates. In addi
tion, the smaller fragments exhibit rapid tumor uptake, are less im
munogenic, and penetrate more deeply into tumors (1—9).
Received 5/4/95; accepted 9/12/95.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
@
One of the clinical disadvantages of radioconjugated antibody
fragments is that they accumulate in the kidneys [sFv > Fab/
Fab' > F(ab')2] to such an extent that they may pose a potential
radiotoxicity problem to this and surrounding organs. Furthermore,
the degree and extent to which kidneys retain radioconjugated MoAbs
depends on which conjugation method is used. Thus, the uptake and
retention of radioiodinated MoAb fragments by certain organs, par
ticularly the kidneys, is much less than those for MoAb fragments
conjugated with radiometal chelates (4, 7, 8). The more rapid decrease
of organ radioactivity after injection of radioiodinated MoAbs is
thought to be due primarily to cleavage of radioiodine by endogenous
dehalogenase enzymes. On the other hand, radiometals such as
I
To
whom
requests
for
reprints
should
be
addressed,
at
Dow
Chemical
Company,
Bioproducts Laboratory, 1701 Building, Midland, MI 48674.
2 Present
3 The
address:
Systemix
abbreviations
used
Corp.,
are:
Palo
MoAb,
Alto,
CA
monoclonal
94304.
antibody;
sFv,
single-chain
Fv;
‘77Lu-CC49,
1―Lu-PA-DOTA-CC49;PA-DOTA; a-[2-(4-aminophenyl) ethylj-1,4,7,10tetraaza-cyclodecane-1,4,7,1O-tetraacetic
acid; SCN-PA-DOTA,
a-[2-(4-isothiocyanato
phenyl) ethyl)-1,4,7,1O-tetraaza-cyclodecane-1,4,7,1O-tetraaceticacid; TBR, total body
retention; AUC, area under the curve; HPLC, high pressure liquid chromatography;
injected dose.
ID,
(10—16).Because of this chemical stability, radionuclidesbound to
i.e., 1@I, 123! or 131!(5, 7, 16—18).
To our knowledge, there is no one method that is currently used on
a routine basis to enhance renal excretion of radioconjugated Fab or
smaller antibody fragments in a clinical setting. There are literature
reports in which large doses of nonradiolabeled antibody have been
administered to test animals and human patients prior to, or in con
junction with, injections of radioconjugated Fab. The results of these
experiments are contradictory. In some cases in which renal uptake of
radioconjugated Fab or F(ab')2 have been decreased, the amount of
“cold―
antibody needed to elicit a desirable effect is extremely large;
therefore, the potential monetary cost and toxicity to the patient make
this approach prohibitive (19—21). Other studies have shown that
charge modification of small proteins can also have a significant
effect on glomerular filtration and renal tubular reabsorption (22, 23).
Published reports that appeared in the 1970s (24, 25) demonstrated
that i.v. injection of certain amino acids and their analogues facilitate
the urinary excretion of various proteins. Mg and Lys were found to
be particularly effective in this regard. These findings were confirmed
by more recent studies, which showed that Lys induced enhanced
excretion of renin protein in mice (26) and of an “
11n-labeled soma
tostatin analogue in man (27). Because amino acids and their ana
logues are small molecules with desirable solubility properties and are
cleared rapidly from the blood, we chose to investigate their potential
utility as drug agents to promote enhanced excretion of Fab from
kidneys. In this study, we report our data using Lys as the renal
clearance enhancement agent and [77Lu]CC49 Fab as the model
radioconjugated antibody fragment. PA-DOTA is a bifunctional che
lating macrocycle that binds ‘77Lu
and other metals of the lanthanide
series (28). CC49, a MoAb that has been characterized extensively
and studied by Schlom and colleagues (29—31),is directed against the
pancarcinoma antigen tumor-associated glycoprotein-72. This antigen
is found on adenocarcinomas of the gastrointestinal tract and in
carcinomas of the breast, ovary, uterus, and non-small cell carcinoma
of the lung (32).
5288
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
LYS AND RENAL EXCRETION OF RADIOCONJUGATED
MATERIALS AND METHODS
radioactivities
Chelation and Activation of SCN-PA-DOTA. To 42 @xl
PA-DOTA (5
mM in H20) were added 250 @xl‘77Lu
solution (1 mp.i in 0.05 N HCI) and 50
pJ 2-(morpholino)ethanesulfonic
acid buffer
was approximately
75%. To activate
the amino
group,
mixer, and the reaction was allowed to continue at room temperature for 10—20
[‘77Lu]SCN-PA-DOTA
reverse-phase
cartridge
was purified
(320-mg
with a 30% acetonitrile:70%
size; Hamilton
on a polyribosyl
Co., Reno,
were
determined.
The remaining
experi
removed, rinsed, blotted dry, and counted.
In the third experiment, groups of mice (n = 3/group)were injected i.p. with
15 p1 thios
phosgene solution (3% in acetonitrile) were added and mixed well on a vortex
mm. The
in liver and kidneys
mental and control groups were anesthetized deeply, followed by intracardiac
perfusion with 15 ml PBS-heparin solution at a flow rate of 5 ml/min. The right
atrium of the heart was severed immediately prior to the initiation of perfusion
to allow drainage of fluid from the circulation. Livers and kidneys were then
(1.0 M; pH 5.8). This was mixed
on a vortex mixer and heated in a 90°Csand bath for 30 mm. The yield of
chelation
MoAb
phosphate
NV), and eluted
water mixture. The yield of [‘77Lu]SCN-PA
50 mg Lys in 1 ml buffer solution at various times (24, 6, 3, 1, 0.5, 0.25, and
0 h) before injection of [‘@LuJCC49
Fab (50 id). Control mice were injected
i.p. with 1 ml PBS. Injection times with PBS were the same as those for the
Lys-injected groups. Fifteen mm after injection of [@Lu]CC49 Fab, groups of
DOTA, based on the starting ‘77Lu
activity, was approximately 70%. The bulk Lys-treated and control mice were sacrificed, and their peritoneal cavities were
of the solvent was dried under a vacuum, and the activated [‘77LuIPA-DOTA blotted dry as described. Blood, liver, and kidneys were collected, weighed@
and placed into counting tubes, and their radioactivities were measured in a
complex in 50 @xl
was used for the subsequent conjugation with antibody.
The CC49 Fab solution containing 3.9 mg/ml protein was exchanged into a
carbonate buffer (50 mM; pH 9.5) using an Amicon concentrator (M, 30,000).
The conjugation
of antibody
fragment
with the radionuclide-chelate
complex
was carried out in carbonate buffer at room temperature. The antibody con
centration was 4 x i0@ M, and the input ratio of the [‘@Lu]SCN-PA-DOTA:
Fab was 0.8 for 80 mm. This resulted in approximately 30% incorporation of
or percentage ID/g.
In the fmal biodistribution experiment, we sought to determine the effect of
a single optimal dose of Lys (50 mg) on the biodistribution of [‘“Lu]CC49
Fab over a 24-h period. For this study, groups of three or four animals first
[‘77LuJSCN-PA-DOTA
Fab conjugate complex was accomplished by gel
received i.p. injections of Lys (50 mg/ml) or PBS. This was followed imme
diately by i.v. injection of [‘@Lu]CC49Fab (50 p1). At various postinjection
times (0.25, 0.5, 1, 3, 6, and 24 h), groups of control and experimental animals
filtration
were sacrificed, and blood, liver, spleen, and kidneys were removed, weighed,
the [‘77Lu]SCN-PA-DOTA into the Fab preparation.
on a PD-b
Pharmacia,
column
Inc., Alameda,
(prepacked
column
Purification
containing
of the
Sephadex
CA), and eluted with PBS. The specific
0-25;
activities
of
and counted as before. The gastrointestinal
the preparations used varied between 1.06 and 1.9 mCi/mI, and the protein
concentration was between 158 and 265 @xWml.Prior to use in biodistribution
studies,
immunoreactivity
was determined
with a tumor-associated
glycoprotein-72
by ELISA in microtiter
wells coated
antigen preparation
as described
Fab. Physical integrity of the radioconjugated
CC49 Fab was examined by
HPLC on a GF-250 Zorbax column (Mac-MOD Analytical, Chaddsford, PA)
and by gel electrophoresis followed by autoradiography. These analyses re
vealed consistently that the final purified product contained <5% aggregates
and was, therefore, an acceptable preparation for use in biodistribution
studies.
Animals. Normalfemale mice of the BALB/c strain(CharlesRiver, Por
tage,
were
were
given
study
MI) were used for these studies. Animals arrived at 28 days of age and
acclimated to the housing conditions for 8—12days before experiments
conducted. Animals were kept on a 12-h light:12-h dark regimen and
free access to rodent chow and water. Throughout the acclimation and
periods,
received
animals
were
prior approval
Biodistribution
treated
according
by the Institutional
Studies.
to study
Animal
Lys (lot L-1262;
protocols
Co., St. Louis,
MO) was dissolved in 10 msi PBS (pH 7.2). f'77LuJCC49Fab was diluted with
the same buffer.
Unless otherwise
noted, the dose of [‘77Lu1CC49 Fab that was
anesthesia.
Thirty
methoxyflurane
mm
later,
all animals
were
methoxyflurane
reanesthetized
inhalation
lightly
mm after [ 77LuJCC49
Fab injections,
animals
with
were anesthe
incision. Peritoneal fluids were blotted dry completely and carefully with
adsorbent pads. Mice were then sacrificed by severing the thoracic aorta just
proximal to the diaphragm. Blood was collected by aspiration with a syringe
and placed into a preweighed tube, weighed, and capped. Liver and paired
kidneys were then removed, rinsed with PBS, blotted dry, and weighed.
Organs were each placed into tubes of appropriate size and counted. Tissue
levels were expressed as percentage ID/organ or as percentage
ID/g.
The second experiment
compartmentalization
in this study was designed to examine the renal
of that fraction
between
corresponding
control
to as TBR.
and experimental
the time course and dose-response
studies, all other
groups
and within
groups
in
studies. Thus, although the number of
animals used in our evaluations may be marginally acceptable from a statistical
perspective,
the fact that the results were duplicated in separate experiments
lends support to our statistically based conclusions.
HPLC of Urine Protein Species. For these studies, groups of mice (n = 3/
group) were each given i.p. injections of Lys at a dose of 50 mg/ml, followed
At 0.25, 0.5, and 1 h after injection, groups of control and experimental mice
were anesthetized and sacrificed as described previously. Urine samples from
animals
were collected
into 1.S-ml conical
polypropylene
tubes as
remaining urine volumes from each animal were pooled according to postin
samples
tized deeply, and the peritoneal cavity was exposed via a ventral midline
radioactivity
and is referred
experiments were repeated on separate occasions to confirm the results re
ported here. The results were similar with respect to relative differences
jection
and given a 50-pJ bolus injection of [t77Lu)CC49 Fab via the
tail vein. Fifteen
animal
With the exception of experiment 2 in the biodistribution
urine was voided by each mouse at the time of sacrifice. A 10-mi sample from
each animal was placed into a separate tube and counted for radioactivity. The
In the first experiment, mice (n = 4/group) received i.p. injections of 0, 10,
while under
in the whole
individual
used in these studies contained between 4.7 and 8.0 @xCiin 0.6—1.1mg protein.
Both reagents were prepared just prior to use.
50, or 100 mg Lys, in 1-mI volumes,
all animal tissues were counted. This allowed us to calculate the percentage ID
15 mm later with an iv. bolus injection of [‘77Lu1CC49Fab (25 pCi/SO
@xl/mouse).As before, control mice were given PBS in place of Lys solution.
that had
Care and Use Committee.
Sigma Chemical
tract and remaining carcass tissues
were divided into eight portions, placed in tubes, and counted for radioactivity,
along with the aforementioned organs and injection standards. The data were
expressed as percentage ID/organ and/or percentage ID/g. In this experiment,
previously (7, 33). By this method, immunoreactivity of the radioconjugated
CC49 Fab was found to be virtually identical to that ofthe unconjugated CC49
@
scintillation counter. The resulting data were expressed as percentage ID/organ
of [‘77Lu]CC49 Fab that is not excreted
in response to Lys treatment. To address this, two groups of mice (n = 3/
group) were each given i.p. injections of PBS containing 50 mg Lys. This was
time and treatment
were frozen
quickly
group.
The three
control
on dry ice and stored
and three
experimental
at —70°C for up to 3 days
prior to HPLC fractionation.
For identification of large-molecular-weight species, urine samples were
thawed and centrifuged
at 5000 X g for 30 mm to remove any sediments.
Volumes of 25—125pi were used for gel filtration HPLC using a DuPont
analytical
GF-250
column.
The mobile
phase was 0.25 M sodium
citrate
(pH
7.0), and the flow rate was 1.5 ml/min. A fraction collector was used to collect
0.25-mm fractions. The radioactivity in each fraction was determined using a
gamma well counter. Pilot studies verified that [‘77Lu]CC49
Fab and the
nonconjugated MoAb had the same column retention times. A purified (cold)
CC49 Fab preparation, therefore, was used as the internal calibration standard
with each sample run. Peaks of interest were cut from the resulting radiochro
matograms and weighed. The remaining portion of each radiochromatogram
was also cut and weighed. The weights that were obtained were used to
followed 30 mm later by iv. injection of [‘77Lu]CC49
Fab. Two groups of
control mice (n = 3/group) were treated with PBS alone. Mice from one
calculate the area under each peak of interest as a percentage of the total AUC.
Statistics. Where appropriate, data were analyzed statistically using stu
dent's I test, one-way ANOVA, or one-way ANOVA followed by the Student
experimental and one control group were each sacrificed 15 mm later, and the
Newman Keuls test.
5289
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
LYS AND RENAL EXCRETION OF RADIOCONJUGATED
RESULTS
@
Results of the first experiment (Fig. 1) show that administration of
increasing doses of Lys resulted in decreased accumulation of radio
activity in kidneys 15 mm after administration of [‘77LuJCC49Fab.
This dose-related effect was statistically significant (F < 0.01). When
compared with mice that were pretreated with 0 mg Lys, treatment
with the 10-mg Lys dose resulted in 27% less accumulation of
radioactivity in kidneys. The intermediate dose of 50 mg decreased
kidney radioactivity 3-fold. Pretreatment with the 100-mg Lys dose
resulted in a 3.6-fold decrease in renal accumulation of radioactivity.
This effect was not significantly different from that obtained with the
50-mg dose. None of the Lys doses had significant effects on radio
activity levels in blood or liver.
The results of the whole-body perfusion experiment are shown in
Table 1. Kidneys from Lys-treated animals retained significantly less
radioactivity than their matched controls (P < 0.05), whereas Lys had
no effect on liver radioactivity levels. Kidneys from both Lys-treated
and control groups that were perfused showed a slight, but significant
(P < 0.05), decrease in radioactivity when compared with the respec
tive nonperfused groups (approximately 24 and 19% less than the
respective controls). However, livers from both perfused groups
showed a 4-fold reduction in radioactivity in comparison to their
respective control groups (P < 0.001). Because the liver is a highly
vascularized organ and receives a major fraction of the cardiac output,
the significant reduction found in the perfused groups indicates that
most of the radioactivity was blood borne. On the other hand, the
kidney data indicate that most of the radioactivity that remained after
perfusion, even after lysine treatment, is probably cell associated.
Data obtained from the third experiment (Table 2) demonstrate that
pretreatment with Lys was effective in decreasing renal accumulation
of the radioimmunoconjugate when the amino acid was administered
up to 6 h before injection of [‘77Lu]CC49Fab. Administration of Lys
at 0.25 h before or concomitant with the radioimmunoconjugate
injection seemed to be the optimal regimen for achieving the greatest
reduction in kidney radioactivity levels (approximately 4-fold). When
Lys was given at 24 h before [‘77Lu]CC49Fab, it proved to be
statistically ineffective in minimizing renal accumulation of radioac
tivity. No significant effects of Lys pretreatment were noted at any of
the time points with regard to radioactivity levels in blood or liver.
Results of the fourth experiment are shown in Figs. 2 and 3. The
140
MoAb
Table 1 Effects of systemic perfusion with buffer solution on kidney and liver contents
of (‘@LuJCC49Fab
micea%lD/gLiver
in control and Lys-treated
KidneysTreatment
LysNo
PBS
perfusion
With
33Caperfusion
Lys
or PBS
control
Lys
6.1 ±0.5
1.4 ±01b
were
injected
PBS
6.7 ±0.7
1.6 ±0.1―
i.p.
30
mm
101.1 ±10.4
82.3 ±0.4c
prior
to iv.
injection
33.4 ±1.4
25.4 ±
of
I'77Lu1CC49
Fab. Mice were sacrificed (no perfusion) or systematically perfused with PBS 15 mm after
injection of [‘77LuJCC49Fab. Data expressed as mean ±SD, n = 3 mice/group.
b
< o.os
and C p < 0.001
when
perfused
versus
nonperfused
treatment
groups
within
the same category are compared.
data show that at all time points examined, kidney retention of
radioactivity was significantly less (P < 0.01) in the Lys-injected
groups relative to their respective time-matched controls (Fig. 2d).
This same dramatic effect was not observed with other organs (Figs.
2, a—c, and 3, a and b) but was evident in all but the 0.25-h
postinjection sampling time in the TBR data (Fig. 3c). It is noteworthy
that the clearance of radioactivity from the carcass (Fig. 3b) was
enhanced slightly by Lys, particularly at the 6-h (1 1.1 ±0.7 versus
14.1 ±0.9%) and 24-h (7.5 ±0.3 versus 9.5 ±0.8%) postinjection
times. It is not known whether this is the result of a specific effect of
Lys on a certain cell type in these tissues or simply the result of a more
rapid clearance rate secondary to renal excretion of radioactivity.
Nevertheless, the combined results of this organ biodistribution cx
periment indicate that enhanced total body clearance of radioactivity
by Lys is mediated primarily, if not exclusively, by the action of the
amino acid on the kidneys.
Results of radioactivity measurements in urine are shown in Table
3. Significantly more radioactivity was excreted by Lys-treated mice
over the first two sampling periods (P < 0.05 versus time-matched
controls). At 1 h after injection, mean counts dropped 2-fold in the
Lys-treated group, whereas they rose in the control group. Conse
quently, the differences at this time point were not statistically dii
ferent. The SDs were large for most of these measurements. This was
not unexpected and probably reflects individual pharmacological re
sponses to handling of the volume load by the kidneys. It is also
probable that from the time of injections to sacrifice, animals voided
a portion of their urine. Thus, the radioactivity levels do not neces
sarily reflect cumulative counts from time of [‘77LuJCC49Fab injec
tion to sampling of urine at time of sacrifice.
The results of size-exclusion chromatography of pooled urine sam
ples are shownin Fig.4. Overthe courseof the samplingperiod,two
major peaks of radioactivity were identified. For purposes of classi
fication, these peaks are designated by the Roman numerals I and II.
In both control and Lys-treated animals, peak I eluted in fractions
100
27—32.This peak coincided with the elution profile for the CC49 Fab
standard. Therefore, it was concluded that peak I most probably
represents intact [‘77Lu]CC49Fab. In both control and experimental
groups, peak I was followed by a lower-molecular-weight
species
eluting in fractions 33—40.This was designated peak I!. Qualitative
examination of peak profiles over time showed clearly that urine
excretion of intact radioimmunoconjugate
increased in the control
group, whereas the opposite occurred in the Lys-treated group. A
second major difference between radiochromatograms of the two
groups was the more dominant appearance of peak II in controls
relative to the experimental group.
0
10
50
100
Quantitative evaluations of the GF-250 radiochromatograms are
Lysine PreTreatment
Dose (mg)
depicted in Table 4. These data show that the sum of areas (calculated
Fig. I . Effects of pretreatment with different doses of Lys on the radiolocalization of
as a percentage of the total AUC in fractions 1—79,inclusive) asso
[177Lu]CC49 Fab in female mice. Mice were given i.p. bolus injections of lysine 30 mm
ciated
with peak I over the 1-h period in the Lys-treated group (182.3)
before iv. administration of (‘77LuICC49 Fab. Mice were sacrificed 15 mm after
were higher than the sum of areas under the corresponding peak in the
1'@LulCC49 Fab injections.
5290
E
I
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
LYS AND RENAL EXCRETION OF RADIOCONJUGATED
Table 2 Effects of i.p.administration
Fob―Interval
MoAb
of PBS or Lys at various times before i.v. injection of (177LuJCC49
between i.p. and
iv. injections
(h)%ID/gBloodLiverKidneysPBSLysPBSLysPBSLys2438.2±4.134.0±2.45.7±1.95.9±1.197.0±5.3111.2±13.1630.6
±335.6
21.6―133.6
13.7―0.536.8
86b0.2532.0
4.2―029.4
a Mice were
sacrificed
b Differences
between
15 mm after injection
corresponding
PBS
±3.824.8
±3.934.7
±0.632.5
±3.24.4
±3.17.0
±3.738.3
±2.46.3
±1.636.5
±4.228.4
±4.34.7
±5.25.5
±4.05.2
of [‘77Lu1CC49 Fab. Data expressed
and
Lys
groups
were
statistically
as mean
significant
±1.897.9
±0.889.5
±3.8108.5
±0.796.9
±9.485.9
±12.949.4
±
±12347.1
±
±20.338.1
±
±
±2.126.4
±20.522.8
±1.1―
ANOVA
followed
by Student-Newman
Keuls
test (P
< 0.05).
DISCUSSION
Several studies have reported recently on the biodistribution prop
erties and potential therapeutic applications of the CC49 MoAb con
jugated with ‘@Lu
via the bifunctional chelating agent PA-DOTA (7,
34, 35). The 177@ isotope is a rare earth metal and was chosen
primarily for its favorable half-life (161 h) and emission spectra. The
half-life is a good match with regard to the tumor biolocalization and
retention properties of various MoAb forms (7, 34, 35). Its emission
15•
Blood
a
±1.2132.7
±SD, n = 3 mice/group.
by one-way
control group (131.0). However, at the 1-h postinjection time alone,
the area under peak I of the Lys group was only slightly higher than
that of its time-matched control. The sum of the areas under peak II
were substantially greater in the control group (50.0) than in the
Lys-treated group (13.9). This was also true when individual time
points were compared. These fractionation experiments indicate that
Lys treatment results in urinary excretion of a larger fraction of intact
[‘77Lu1CC49Fab (peak I) compared with that in vehicle-injected
controls.
50
±1.4108.2
±2.04.1
±1.06.4
±1.06.4
±1.37.2
±0.96.9
±0.45.8
b
Spleen
----@---FBS
12@
@40.
—0-—
LYS
—C—-
LYS
9.
6@
3@
Li::+
—
@
U-
0
10
4
8
12
16
20
24
Liver
C
350300-
@
@
@
II
8
I''l'''U'''
0
—0—
4
8
d
12
U
16
20
24
Kidneys
----U---
P135
—a-— LYS
LYS
250@
6
2ø3@
150.@
4
100
50@
@
0@ @w
•
0
0
4
8
12
Post-Injection
16
20
24
0
Time (firs)
.
4
U
8
12
Post-Inj@tion
16
2D
24
Time (Hrs)
Fig. 2. Time course study of the radiolocalization and clearance of l'77Lu1CC49 Fab from: a, blood; b, spleen; c, liver; and d, kidneys of female mice injected i.p. with Lys.
[‘77Lu]CC49
Fab was injected iv. Both bolus injections were given at time 0.
5291
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
LYS AND RENAL EXCRETION OF RADIOCONJUGATED
a
G.I. Tract
MoAb
prolonged
retentioninkidneysthatweandothershaveobservedwhen
using antibody fragments conjugated
- - - -..
I
I
- -
PBS
Because
—a--— LYS
4
2
0
40
8
b
12
16
20
24
Carcass
----@---PBS
I
I
—v----
30
LYS
0
0
4
8
12
I
I
16
20
----U---
PBS
—0-—-•
LYS
24
radioactivity
0
0
4
8
12
16
20
radiotoxicity
to metal-chelate
may
result
from
complexes.
renal
accu
examine potential pharmacological approaches to reduce renal accu
mulation of these small-molecule radiopharmaceuticals. If successful,
MoAb fragments and genetically engineered constructs such as sFv
forms may become even more attractive for diagnosis and/or therapy
of tumors, particularly for micrometastatic foci that are located within
the peritoneal cavity in the vicinity of the renal excretory organs.
The results of our studies indicate clearly that the renal excretion
rate of [‘77Lu]CC49Fab is enhanced significantly by i.p. administra
tion of Lys. We found that a single injection of 50 mg/mouse was the
optimal dose with which to exert a significant stimulatory effect on
urinary excretion of radioactivity. In preliminary range-finding exper
iments, we observed that Lys doses of 150 mg or more led to lethargic
behavior in most animals and death in a smaller number of others. No
such adverse responses were noted in any of the animals injected with
up to 100 mg Lys. Because the desirable renal effect with the 50-mg
dose was not substantially different from that of the 100-mg dose, we
chose to conduct the rest of the experiments using the smaller dose.
Our studies suggest that the renal effects of Lys are most pronounced
when it is administered within a specific window of time before, or
concomitant with, i.v. injection of the radioimmunoconjugate. This
effect of Lys is extremely rapid in onset, a finding that corroborates
similar actions of this amino acid on urinary excretion of f32-micro
globulin in man (25). In fact, this latter study found that large amounts
of the protein appeared in urine simultaneously with [‘@I1iodo
thalamate, a filtration marker that is not reabsorbed by the tubules
(37). In our study, we found a 3-fold reduction in kidney radioactivity
within 15 mm after coadministration of Lys and [‘77Lu]CC49Fab
(Fig. 2d). At 6 h after injection, this difference was close to 6-fold and
was due almost entirely to the continued accumulation of the radio
immunoconjugate in kidneys of control mice. In contrast, Lys-treated
animals showed only a modest increase in renal accumulation of
6
4
organ
mulationof radioconjugated
CC49MoAb fragments,we soughtto
8
0
significant
24
Post-Injection r@e (firs)
Fig. 3. Time course of the radiolocalization and clearance of (‘“LuJCC49
Fab from:
a, the gastrointestinal tract; b, carcass; and c, TBR of female mice coinjected with Lys.
Note that the ordinate units reflect the total amount of activity in tissues without
correction for their weight.
over
this
same
period
of time.
Systemic perfusion of animals injected with [‘77Lu]CC49Fab
resulted in qualitatively similar effects on the liver and kidneys; that
is, washout of blood from the vascular compartment with perfusion
buffer resulted in significantly lower concentrations of radioactivity in
both organs. However, the quantitative effects of perfusion in the two
organs were quite different. This implies that the mode of radioim
munoconjugate metabolism in kidney and liver are different. The low
levels of radioactivity that remained after perfusion in the liver may
reflect entrapment of the radioimmunoconjugate by hepatic cells. This
is supported by the autoradiography studies of Yokota et a!. (38).
These investigators found that radioiodinated CC49 MoAb fragments
and sFv localized to Kupfer cells in tumor-bearing mice, although the
density of silver grains was much lower than for the IgG form.
Furthermore, results of our 24-h biodistribution study show that liver
radioactivity levels are highest at 15 mm after injection, after which
Table 3 Concentrations of radioactivity in urine of mice after i.p. administration of
spectra include a f3 component, which may be useful for tumor
therapy, and a @y
component, which is conducive to radiodiagnoses
with a ‘y
camera or hand-held y detection probe (34, 36). In-depth
discussions of the pharmacological properties and potential diagnostic
and therapeutic utilities of CC49 MoAb forms conjugated with the
[‘77Lu]PA-DOTA complex can be found in several publications by
Schlom and coworkers (7, 34, 35). Our interest was to use the
[177Lu]CC49 Fab radioimmunoconjugate
as a model system with
which to investigate the pharmacological basis for the high and
PBS or Lvs and i.v. injection of I 77LuJCC49
Fab―c@10
@d
Postinjection
time
Lysine0.25
(h)
PBS
2258b0.5
289.5―I
189.0 ±82.0
65.5 ±0.7
316.0±45.3a
Fab.Data
Mice were injected
715.7 ±
740.0 ±
245.0±80.8
with PBS or Lys 15 mm prior to injection
with [‘77Lu]CC49
expressed as mean ±SD, n = 3 mice/group.
significantby
b Differences
between
corresponding
PBS
and Lys groups
were
statistically
one-way ANOVA followed by Student-Newman Keuls test (P < 0.05).
5292
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
LYS AND RENAL EXCRE11ON OF RADIOCONJUGATED
70000
CC49 Fab
4@
50000
CC49 and its sFv form were filtered through the glomerulus and
accumulated in cortical tubules. The smallest MoAb form showed the
most rapid tubular accumulation and subsequent clearance from these
structures. They postulated that the rapid diminution of renal radio
activity was the result of local dehalogenation and subsequent excre
tion of radioiodine into the urine. Because our radioisotope was
conjugated to the MoAb fragment via a bifunctional chelate, the
HRS
60000
PBS
‘10@25
Lys
40000
b 30000
radioimmunoconjugateis more resistantto catabolismby tissue en
zymes. Consequently, radioactivity concentrations in the kidney were
much higher and of longer duration over the course of the experiment.
Similar observations have been made by other workers using this (7,
34) and other radioactive metal-chelate complexes (4, 8, 39). The fact
that we were unable to effect a more pronounced washout of kidney
radioactivity by systemic perfusion in either PBS- or Lys-treated
animals further suggests that radionuclide-coupled species of the
radioimmunoconjugate are bound tightly to membrane and/or cellular
components of tubular cells. Sites of binding to tubular cells for these
molecules, and their binding constants, should be determined to obtain
a better understanding of their cellular pharmacological properties.
The exquisite renal specificity and overall impact of Lys on total
radioactivity levels is borne out in the TBR data (Fig. 3c). These data
show that nearly all of the difference in the cumulative 24-h TBR
between the control and experimental groups was accounted for by
radioactivity found in the kidneys. To quantify differences in the TBR
between control and Lys-treated groups, the relative AUCS were
determined for the time period between 0.25 and 24 h after injection.
The AUC for the control group was calculated to be 263, and that for
the Lys-treated group was 104. A single i.p. dose of Lys was, there
fore, able to reduce the TBR of radioactivity by a factor of more than
2.5-fold. It is conceivable, although not yet tested, that multiple
injections or infusion of the amino acid would enhance the renal
excretion of radioactivity further. This conjecture is supported by the
results of early studies by Mogensen et a!. (24) regarding the effects
of amino acid infusions on urinary excretion of proteins in healthy
male volunteers. In their initial study, the investigators found that i.v.
administration of increasing doses of Arg (3, 6, 9, and 12 g) for 2 mm
each resulted in significant enhancement of urinary excretion of
immunoglobulin light chains and @2-microglobulin,both of which are
freely filtered by the glomerulus. After infusion of the 12-g dose,
urinary excretion oflight chains increased from a baseline of 5.5 to 52
@gimin,and that for @32-microglobulinincreased from 0.092 to 25.71
@Wmin.In their follow-up study (25), it was found that Lys was even
more potent than Arg. This was especially true for @2-microglobulin,
the smallest protein studied (@@Mr12,000), which was excreted at a
rate that was —1,600times above the pre-Lys infusion baseline.
Results of urine quantitation of radioactivity and HPLC analyses of
the remaining pooled samples indicate that Lys treatment diminishes
the renal catabolism of [1@Lu]CC49 Fab, at least during the early
20000
10000
0
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
70000
60000
50000
@
40000
U
30000
20000
10000
0
70000
60000
50000
@
40000
U
MoAb
30000
20000
10000
0
FRACTION
NUMBER
stages of biolocalization to the kidney. In fact, the area underpeak I,
Fig. 4. Gel filtration (GF-250) HPLC radiochromatograms of pooled urine samples at
three different postinjection times. Tracings represent pooled samples from control (PBS)
1:@nme@@t5l(Lys) groups. Quantitative analyses of peaks I and II are shown in
which represents
presumably
a nonmetabolized
radioimmunoconju
gate, was 41% greater in Lys-treated
animals than in controls. It is
highly probable, therefore, that at postinjection
times preceding
15
mm, the difference in urine concentration
they fall rapidly. This clearance pattern, along with our liver perfusion
results, supports the hypothesis that at 15 mm after injection of
[‘77Lu]CC49Fab, most of the liver radioactivity is confined to the
sinusoidal compartment. The kidney data, on the other hand, indicate
that only a small, albeit significant, fraction of radioactivity is found
in the renal blood pool. That reabsorption of MoAb fragments occurs
at cortical tubular sites has been demonstrated elegantly by Yokota et
a!. (38) with the aid of tissue autoradiography techniques. In their
study, they showed that the F(ab')2 and Fab' proteolytic fragments of
of radioactivity between
samples―PostinjectionPBSLysinetime(h)IIIIII0.2535.219.760.25.90.550.314.363.04.2145.516.059.13.8
Table 4 Gelfiltration (G-250) HPLC analyses of urine
a Details
regarding
data reduction
are explained
in “Materials and Methods.― Total
areas used in the analyses were between fractions 1—79,
inclusive. Areas under designated
peaks are expressed as percentage of total area under the curve.
5293
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
LYS AND RENAL EXCRETION OF RADIOCONJUGATED
PBS- and Lys-treated animals would be even greater. These data
may be indicative of saturation of tubular cells by Lys, which then
minimizes the number of free binding sites available to filtered
[‘77Lu]CC49Fab greatly. Consequently, its shorter residence time
in kidneys of animals treated with Lys leads to urinary excretion of
a high percentage of intact radioimmunoconjugate
and a signifi
cantly smaller percentage of degradation products. In the controls,
on the other hand, more free tubular sites are available to the
[‘77Lu]CC49Fab, thereby leading to a greater number of radioim
munoconjugate molecules binding to the lumenal surface of tubu
lar cells where they undergo hydrolysis by cellular brush border
peptidases (40).
The mechanism by which Lys exerts its stimulatory effect on renal
excretion of protein is not understood fully. The studies of Mogenson
and Solling (25) demonstrated that, as a group, the dibasic amino
acids Lys, Arg, and ornithine exerted the most potent effects on
urinary protein excretion. Amino acids lacking a terminal amino
group or terminal groups that were not positively charged had no renal
effect. These and other (41, 42) findings have led to the hypothesis
that positively charged amino acids bind to negatively charged sites
on the intralumenal surface of renal tubular cells and thereby block
tubular binding of free, positively charged amino acid residues that are
present on peptide backbones of freely filtered proteins. Although
electrostatic forces between amino acids and tubular cells play a
significant role in protein excretion, it does not explain why Lys was
found to be significantly more potent than other similar amino acids
or their analogues. For example, 6-fold more /32-microglobulin was
excreted on infusion of Lys when compared with the amino acid
analogue c-amino caproic acid, although the latter differs structurally
from Lys in that it does not possess an a-amino group. Based on this
and other structure-activity considerations, Mogenson and Solling
(25) suggested
that the potent
renal effect
of Lys must be due to its
MoAb
ACKNOWLEDGMENTS
We thank Dr. Jeffrey Schlom (National Cancer Institute, Bethesda, MD) for
providing
us with the CC49 cell line. We are grateful to 0. Spittka and
C. Harrington for their excellent technical assistance in conducting this study.
REFERENCES
1. CovelI, D. G., Barbet, J., Holton, 0. D., Black, C. D. V., Parker, R. J., and Weinstein,
J. N. Pharmacokinetics of monoclonal immunoglobulin 0,, F(ab')2 and Fab' in mice.
Cancer Res., 46: 3969—3978,1986.
2. Holton, 0. D., III, Black, C. D. V., Parker, R. J., Covell, D. G., Barbet, J., Sieber,
S. M., Talley, M. J., and Weinstein, J. N. Biodistribution of monoclonal IgG,, F(ab')2
and Fab' in mice after intravenous injection: comparison between anti-B cell (anti
LyB8.2) and irrelevant (MOPC-21) antibodies. J. Immunol., 139: 3041—3049,1987.
3. Fujimori, K., Covell, D. G., Fletcher, J. E., and Weinstein, J. N. Modeling analysis of
the global and microscopic distribution of immunoglobulin G, F(ab')2 and Fab in
tumors. Cancer Rca., 49: 5656—5663,1989.
4. Goldenberg, D. M., Goldenberg, H., Sharkey, R. M., Higginbotham-Ford,
E., Lee,
R. E., Swayne, L C., Burger, K. A., Tsai, D., Horowitz, J. A., Hall, T. C., Pinsky,
C. M., and Hansen, H. J. Clinical studies of cancer radioimmunodetection with
carcinoembryonic antigen monoclonal antibody fragments labeled with ‘@I
or
Cancer Res., 50 (Suppl.): 909s—921s,1990.
5. Carrasquillo, J. A., Krohn, K. A., Beaumier, P., McGuffin, R. W., Brown, J. P.,
Hellstrom, K. E., and Larson, S. M. Diagnosis of and therapy for solid tumors with
radiolabeled antibodies and immune fragments. Cancer Treat. Rep., 68: 317—328,
1984.
6. Halpern, S. E., and Dillman, R. 0. Problems associated with radioimmunodetection
and possibilities for future solutions. J. Biol. Response Modif., 6: 235—262,
1987.
7. Schott, M. E., Milenic, D. E., Yokota, T., Whitlow, M., Wood, J. F., Fordyce, W. A.,
Cheng, R. C., and Schlom, J. Differential metabolic patterns of iodinated versus
radiometal chelated anticarcinoma single-chain Fv molecules. Cancer Res., 52: 6413—
6417, 1992.
8. Brown, B. A., Comeau, R. D., Jones, P. L., Liberatore, F. A., Neacy, W. P., Sands,
H., and Gallagher, B. M. Pharmacokinetics of the monoclonal antibody B72.3 and its
fragments labeled with either ‘@I
or “In.Cancer Rca., 47: 1149—1154, 1987.
9. Yokota, T., Milenic, D. E., Whitlow, M., and Schlom, J. Rapid tumor penetration of
a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res., 52:
3402—3408, 1992.
10. Sundberg, M. W., Meares, C. F., Goodwin, D. A., and Diamanti, C. I. Chelating
agents for the binding of metal ions to macromolecules.
587—588,1974.
Nature (Land.), 250:
11. Griffin, T. W., and Doherty, P. W. Radioactive labeling of antibody: a simple and
efficient method. Science (Washington DC), 220: 613—615, 1983.
binding to a receptor system on the tubular cell. Whether this receptor
is distinct from the putative receptors for other amino acids is not clear
(41).
These studies demonstrate that the renal excretion rate of
[‘77Lu]CC49Fab is enhanced significantly by preadministration of
Lys within a specific window of time prior to injection of the
radioimmunoconjugate.
This Lys effect is dose dependent and
rapid in onset, as evidenced by the significant reduction of renal
radioactivity levels when the amino acid is given just prior to
[‘77LuJCC49Fab. Additionally, this Lys effect seems to be medi
ated via blockade of tubular binding sites for the radioimmuno
conjugate, as evidenced by data obtained on fractionation of urine
using gel filtration HPLC.
After completion of our studies, Behr et a!. (43) corroborated and
extended our findings. These investigators reported animal model and
clinical data with regard to the effects of Lys on renal and urine
radioactivity levels after administration of a [99mTcJFab1 radioimmu
noconjugate. They also reported that a range of basic amino acid
derivatives, including the D-isomer of Lys and Lys polymers, were
effective in reducing renal radioactivity to varying degrees. Because
their animal studies were carried out using mice bearing human colon
carcinoma xenografts, they were also able to show that Lys did not
affect tumor uptake oftheir radioconjugated Fab'. These findings lend
support to our contention that Lys or its derivatives should be exam
med further as potentially useful agents for reducing accumulation of
radioactive metal-chelate-Fab (or smaller MoAb forms) complexes in
kidneys. This may not only reduce potential renal radiotoxicity, but
also aid in improved diagnoses and/or therapy of tumors that form
within structures of the peritoneal region.
12. Meares, C. F., McCall, M. J., Reardan, D. T., Diamanti, C. I., and McTigue, M.
Conjugation of antibodies with bifunctional chelating agents: isothiocyanate and
bromoacetemide reagents, methods of analysis, and subsequent addition of metal
ions. Anal. Biochem., 142: 68—78,1984.
13. Hnatowich, D. J., Virzi, F., and Doherty, P. W. DTPA-coupled antibodies labeled
with yttrium-90. J. Nucl. Med., 26: 503-509, 1985.
14. Moi, M. K., Meares, C. F., McCall, M. J., Cole, W. C., and DeNardo, S. J. Copper
chelates as probes of biological systems: stable copper complexes with a macrocyclic
bifunctional chelating agent. Anal. Biochem., 148: 249—253, 1989.
15. Moi, M. K., DeNardo, S. J., and Meares, C. F. Stable bifunctional chelates of metals
used in radiotherapy. Cancer Res., 50 (Suppl.): 789s—793s, 1990.
16. Halpem, S. E., Hagan, P. L, Garver, P. R., Koziol, J. A., Chen, A. W. N., Frincke,
J. M., Bartholomew, R. M., David, G. S., and Adams, T. H. Stability, characterization,
and kinetics of “In-labeledmonoclonal antitumor antibodies in normal animals and
nude mouse-human tumor models. Cancer Res., 43: 5347—5355,1983.
17. Buchsbaum, D., Randall, B., Hanna, D., Chandler, R., Loken, M., and Johnson, E.
Comparison of the distribution and binding of monoclonal antibodies labeled with
131-iodine or 111-indium. J. Nucl. Med., 10: 398—402,1985.
18. Sharkey, R. M., Motta-Hennessy,
C., Pawlyk, D., Siegel, J. A., and Goldenberg,
D. M. Biodistribution and radiation dose estimates for yttrium- and iodine-labeled
monoclonal antibody IgG and fragments in nude mice bearing human colonic tumor
xenografts. Cancer Res., 50: 2330—2336,1990.
19. Boerman, 0. C., Blumenthal, R. D., Sharkey, R. M., Fand, I., Aninipot, R., and
Goldenberg, D. M. The influence of antibody protein dose on the biodistribution,
tumor penetration, and therapeutic efficacy of radioiodinated antibodies in the
GW-39/nude mouse model. Proc. Am. Assoc. Cancer Res., 32: 261, 1991.
20. Kennel, S. J., Falcioni, R., and Wesley, J. W. Microdistribution of specific rat
monoclonal antibodies to mouse tissues and human tumor xenografts. Cancer Res.,
51: 1529—1536,
1991.
21. Buchsbaum, D. J., Wahl, R. L., Glenn, S. D., Normolle, D. P., and Kaminski, M. S.
Improved delivery of radiolabeled anti-Bi monoclonal antibody to Raji lymphoma
xenografts by predosing with unlabeled anti-Bi monoclonal antibody. Cancer Res.,
52: 637—642,1992.
22. Rennke, H. G., Patel, Y., and Venkatachalam, M. A. Glomerular filtration of proteins:
clearance of anionic, neutral, and cationic horseradish peroxidase in the rat. Kidney
Int., 13: 278—288,1978.
23. Tarburton, J. P., Halpern, S. E., Hagan, P. L, Sudora, E., Chen, A., Fridman, D. M.,
and Pfaff, A. E. Effect of acetylation on monoclonal antibody ZCE-025 Fab':
distribution in normal and tumor-bearing mice. J. Biol. Response Modif., 9: 221—230,
1990.
5294
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
LYSAND RENALEXCRETIONOF RADIOCONJUGATED
MoAb
24. Mogensen, C. E., Vittinghus, E., and Solling, K. Increased urinary excretion of
albumin, light chains, and @3@-microglobulinafter intravenous arginine administration
in normal man. Lancet, 2: 581—583,1975.
25. Mogensen, C. E., and Solling, K. Studies on renal tubular protein reabsorption: partial
and near complete inhibition by certain amino acids. Scand. J. Gin. Lab. Invest. 37:
477—486,
1977.
26. Mazanti, I., Hermann, K. L., Nielsen, A. H., and Poulsen, K. Ultrafiltration of renin
in the mouse kidney studied by inhibition of tubular protein reabsorption with lysine.
Clin. Sci. (Land.), 75: 331—336,1988.
27. Hammond, P. J., Wade, A. F., Gwilliam, M. E., Peters, A. M., Myers, M. J.,
Gilbey, S. G., Bloom, S. R., and Calam, J. Amino acid infusion blocks renal
tubular uptake of an indium-labeled somatostatin analogue. Br. J. Cancer, 67:
1437—1439,
1993.
28. Cheng, R. C., Fordyce, W. A., Frank, J. R., Goeckeler, W. F., Kiefer, G., Kruper,
34. Schott, M. E., Schlom, J., Siler, K., Milenic, D. E., Eggensperger, D., Colcher, D.,
Cheng, R., Kruper, W. J., Jr., Fordyce, W., and Goeckeler, W. Biodistribution and
W. J., Jr., McMillan, J. S., Simon, J. S., Wilson, D. A., and Baughman, S. Dow
European Patent Application No. 353,450.
38. Yokota, T., Milenic, D. E., Whitlow, M., Wood, J. F., Hubert, S. L, and Seldom, J.
29. Johnson, V. G., Schlom, J., Paterson, A. J., Bennett, J., Magnani, J. L., and Colcher,
D. Analysis of a human tumor-associated glycoprotein (TAG-72) identified by a
monoclonal antibody B72.3. Cancer Res., 46: 850—857,1986.
30. Sheer, D. G., Schlom, J., and Cooper, H. L. Purification and composition of the
human tumor-associated glycoprotein (TAG-72) defined by monoclonal antibodies
CC49and B72.3.CancerRes.,48: 6811—6818,
1988.
31. Kuroki, M., Fernsten, P. D., Wunderlich, D., Colcher, D., Simpson, J. F., Poole, D. J.,
and Schlom, J. Serologic mapping of the TAG-72 tumor-associated antigen employ
ing 19 distinct monoclonal antibodies. Cancer Res., 50: 4872—4879, 1990.
32. Molinolo, A., Simpson, J. F., Thor, A., and Schlom, J. Enhanced tumor binding using
immunohistochemical analyses by second generation anti-tumor associated glycopro
tein 72 monoclonal antibodies versus monoclonal antibody B72.3 in human tissue.
Cancer Res., 50: 1291—1298,1990.
33. Milenic, D. E., Yokota, T., Filpula, D. R., Finkelman, M. A. J., Dodd, S. W., Wood,
J. F., Whitlow, M., Snoy, P., and Schlom, J. Construction, binding properties,
preclinical radioimmunotherapy studies using radiolanthanide-labeled immunoconju
gates. Cancer (Phila.), 73 (Suppl.): 993—998,1994.
35. Schlom, J., Siler, K., Milenic, D. E., Eggensperger, D., Colcher, D., Miller, L S.,
Houchens, D., Cheng, R., Kaplan, D., and Goeckeler, W. Monoclonal antibody-based
therapy of a human tumor xenograft with a 177lutetium-labeled immunoconjugate.
Cancer Res., 51: 2889—2896, 1991.
36. Sickle-Santanello, B. J., O'Dwyer, P. J., Mojzisik, C., Thule, S. E., Hinkle, G. H.,
Rousseau, M., Schlom, J., Colcher, D., Thurston, M. 0., Nieroda, C., Sardi., A.,
Minton, J. P., and Martin, E. W. Radioimmunoguided surgery using the monoclonal
antibody B72.3 in colorectal tumors. Dis. Colon Rectum, 30: 761—764,1987.
37. Mogensen, C. E. Glomerular filtration rate and renal plasma flow in short-term and
long-term juvenile diabetes mellitus. Scand. J. Clin. Lab. Invest., 28: 91—100,1971.
Microautoradiographic analysis of the normal organ distribution of radioiodinated
single-chain Fv and other immunoglobulin forms. Cancer Res., 53: 3776—3783, 1993.
39. Yorke, E. D., Beaumier, P. L, Weasels, B. W., Fritzberg, A. R., and Morgan, A. C.,
Jr. Optimal antibody-radionuclide combinations for clinical radioimmunotherapy: a
predictive model based on mouse pharmacokinetics. mt. J. Radiat. Appl. Instrum. Part
B, 18:827-835,1991.
40. Nielsen, J. T., Nielsen, S., and Christensen, E. 1. Transtubular transport of proteins in
rabbit proximal tubules. J. Ultrastruct. Res., 92: 133—145,
1985.
41. Silbernagel, S., Pfaller, W., and Deetjen, P. Molecular specificity of tubular amino
acid reabsorption. in: Current Problems in Clinical Biochemistry, Vol. 6, pp. 403—
415. Bern, Switzerland: Hans Huber Publishers, 1976.
42. Pfaller, W., and Silbernagl, S. Cellular localization of L-Arginine reabsorption in
proximal tubules ofrat kidney cortex. Pfluegers Arch. Eur. J. Physiol., 360: 189—192,
1975.
43. Behr, T. M., Sharkey, R. M., Juweid, M. E., Aninipot, R., and Goldenberg, D. M.
Reduction of kidney uptake of Fab' fragments of monoclonal antibodies: animal
experiments and initial clinical results. Proc. Am. Assoc. Cancer Res., 36: 617,
metabolism, and tumor targeting of a single-chain Fv derived from the pancarcinoma
monoclonal antibody CC49. Cancer Res., 51: 6363—6371, 1991.
1995.
5295
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
Lysine Reduces Renal Accumulation of Radioactivity
Associated with Injection of the [ 177Lu]α-[2-(4-Aminophenyl)
ethyl]-1,4,7,10-tetraaza-cyclodecane-1,4,7,10-tetraacetic
acid-CC49 Fab Radioimmunoconjugate
Louis R. DePalatis, Kevin A. Frazier, Roberta C. Cheng, et al.
Cancer Res 1995;55:5288-5295.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/55/22/5288
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.