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Dual Isotope Study of Iodine-125 and Indium-111-Labeled Antibody in Athymic Mice
Patrick L. Carney, Patrick E. Rogers and David K. Johnson
J Nucl Med. 1989;30:374-384.
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(Print ISSN: 0161-5505, Online ISSN: 2159-662X)
© Copyright 1989 SNMMI; all rights reserved.
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Dual Isotope Study of Iodine- 125
and Indium-i 11-Labeled Antibody
in Athymic Mice
Patrick L. Carney, Patrick E. Rogers, and David K. Johnson
Abbott Laboratories, Department 90M, Abbott Park, illinois
MonoclonalantibodyB72.3was coupledto a benzylisothiocyanate
derivativeof
diethylenetnaminepentaacetic
acid(DTPA).The maximumsubstitutionachievablewithoutloss
of immunoreactivity was three DTPA groups per immunoglobulin molecule. The resulting
conjugatewas labeledwith 111Inby briefincubationwith 111lnCl3,
givinga mean radiochemical
yieldof 111In-Iabeled
antibodyof 96%.The [‘11ln]B72.3
preparationwas mixedwith an [1251]
B72.3 preparation, obtained by the chloramine-Tmethod, and the mixture administered to
athymicmicebearingsubcutaneousLS174T coloncarcinomaxenografts.Therewereno
significantdifferences(p > 0.1)in the bmodistnbutions
of the two labelsat 1, 2, 5, and7 days
postinjection.Theseresultsarecontrastedwith prior studiesshowingelevatedlevelsof 111In
in liver,spleen,and kidneysusingB72.3-DTPAconjugatespreparedvia the bicyclic
anhydnde.It is concludedthat proteincross-linkingand/orthe formationof unstablechelate
sitesin anhydridecoupledconjugatesunderliethesedisparities.
J Nucl Med 30:374—384,1989
diolabeled immunoglobulins show considerable
promise as a means oftumor detection and, potentially,
tumor therapy (1—3).Initial work with radioiodinated
polyclonal antisera (4,5) and, more recently, with io
dine-13l- (‘@‘I)
and iodine-123- (1231)labeled mono
clonal antibodies has clearly demonstrated immunolog
ically mediated tumor targeting in animal models and
in a number of human clinical trials (6-12). For diag
nostic applications, ‘@‘I
is suboptimal due to the high
beta dose delivered to tissues such as the thyroid and
bladder, the poor resolution of gamma camera images
as a result of the relatively high-energy emissions from
this radionuclide and because of a tendency for the
iodine label to be cleaved from the immunoglobulin in
vivo through poorly understood dehalogenation mech
and other organs ofthe reticuloendothelial system (14—
16,22-24). This constitutes a severe limitation on the
clinical utility of such agents, as many tumors tend to
metastasize to the liver. The origins ofthis tendency for
“In-labeled
antibodies to accumulate in the liver to a
greater extent than do radioiodine-labeled antibodies
are unclear. At least three distinct mechanisms can be
invoked to account for the observed behavior of these
labeled immunoglobulins.
The first hypothesis is that antibodies themselves are
taken up by the liver, either through binding to Fc and!
or carbohydrate receptors present on liver cells, or as a
result of the formation of antibody-antigen complexes
when the target antigen is present in the circulation. It
can then be argued that indium- and iodine-labeled
anisms. For these reasons, indium-l 11 (‘
‘
‘In),a
gamma-emitting
radiometal with photon energies of
173 keY and 247 keV, has been widely used as an alter
antibodies are both taken up by the liver to the same
extent but that, subsequently, the iodine-labeled anti
labeling being achieved by covalent modification of the
antibody molecule with chelating moieties (17—21).
thyroid, stomach, and urine. Indium, on the other hand,
is retained in the liver either by virtue of remaining
bodies undergo dehalogenation reactions leading to re
native label for monoclonal antibody studies (13—16), lease of the label from the liver and redistribution to
A key disadvantage to date with ‘
‘
‘In-labeledanti
bound in stable fashion to the antibody-chelator con
bodies has been high uptake of this label into the liver
jugate or, if released from the chelate, by entering the
metabolic pathways involved in iron storage.
ReceivedMay 12, 1988;revision accepted Oct. 11, 1988.
Forreprintscontact:DavidK. Johnson,PhD, AbbottLabora
tories, D9OM,Abbott Park, IL 60064.
374
Camey,Rogers,andJohnson
A second mechanism that could account for elevated
liver levels of indium involves changes in the integrity
of the IgG molecule resulting from the conditions em
TheJournalof NuclearMedicine
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ployed in labeling. Were the derivatization of an anti
body with chelating groups to lead to greater denatu
ration of that antibody than does the corresponding
adenocarcinomas (29).
halogenation
MATERIALS AND METhODS
reaction, it would be anticipated
that the
more denatured material would be scavenged by the
reticuloendotheial
system to a greater extent, leading
bound by transferrinand processed through the path
Antibody Purification
The hybndoma producing B72.3 (American Type Culture
Collection, Rockville, MD) was grown in tissue culture using
an airlift fermenter. The antibody was purified from the
culture medium by affinity chromatography on protein A
Sepharose CL-4B (Repligen Corp., Cambridge, MA), followed
ways by which the body regulates iron metabolism.
by exhaustivedialysisagainstPBS.For the chelatecoupling
to higher liver levels of indium than of iodine.
The third mechanism relates to instability of the
indium chelate label. If the chelate complex were to
dissociate
in circulation,
free indium(III)
would be
As
a major pathway entails transferrin-mediateduptake of
the metal into liver parenchyma and subsequent depo
sition in the iron storage protein, ferritin,such instabil
ity would be expected to produce elevated levels of
indium in the liver.
Preparation of Antibody-Chelator Conjugates
The isothiocyanatederivativeofDTPA (Fig. 1),the synthe
of the iodine-tyrosine bond in vivo would be the pri
sis of which will be described elsewhere (Westerberg DA,
Carney PL, Rogers PE, et al. J. Med. Chem: in press), was
dissolved in a minimum volume of 0.1M KH2PO4/0.1M
NaHCO3, pH 8.5, and added to a solution of the antibody in
the same buffer at a concentration of 10 mg/mi. The resulting
solution was incubated at 37°Cfor 3 hr then was dialysed at
radioactivity
seen with these two labels and changes in
the chelate coupling method and in chelate stability
would not be expected to greatlyalter these patterns. If,
on the other hand, the latter two mechanisms were to
be important then the particularchelate labeling meth
odology and the nature of the chelate label would be
expected to influence relative liver levels of iodine and
indium activity. As the overwhelming majority of stud
ies to date have employed anhydride coupling of die
thylenetriaminepentaacetic acid (DTPA) as the conju
gation method and the resulting diethylenetriaminete
traacetic acid (DTTA) residue as the chelator, we sought
to evaluate the effects that a different coupling method
and chelate would have on relative liver levels of the
two labels. We therefore undertook an ‘
‘
‘In/'25I
dual
isotope study using a benzylisothiocyanate derivative of
diethylenetriaminepentaacetic
acid (DTPA) (Fig. 1) re
cently prepared in this laboratory (25) and B72.3, an
IgG,
murine
HO2C
\
monoclonal
antibody
developed
r—\
,/—
by
CO2H
N
@
procedure, the antibody was dialyzed into 0.1M KH2PO4/
0.1M NaHCO3, pH 8.5. Antibody concentrations were mess
ured by the Bradford dye binding assay(Bio Rad Laboratories,
Richmond, CA).
If the first hypothesis is correct, then the instability
mary factor underlying the observed patterns of liver
@
Schlom et al. (26—28)that is reactive with a variety of
HO2C
CO2H
CO2H
2—8°C,
first against a 0.lMsolution
ofDTPA in 0.05M citrate
buffer,pH 6, for 24—48
hr then against0.05M citrate buffer,
pH 6, for a further3 days. If necessary,the antibody concen
tration was readjusted to 10 mg/ml then the conjugate solu
tion wasaliquotedinto acidwashedglassvialsand storedat
2—8°C
until needed. The average number of chelating groups
bound to each antibody molecule was determined by a thin
layer chromatography (TLC) cobalt-Si binding assay previ
ously described by Meares et al. (30).
Labeling of Antibody-Chelator Conjugates with Indium-ill
Carrifree'
‘In]chloride(AtomicEnergy ofCanada, Ltd.,
Kanata, Ontario) (1.0 mCi) was added to 100 @ilof the
antibody-chelator conjugate at a concentration of 10 mg/ml
in 0.05M citrate buffer, pH 6. After 30 mm incubation at
room temperature, the radiochemical yield of “In-labeled
B72.3 was determined by performing a DTPA chase to se
quester that fraction of added ‘
‘
‘Innot taken up by the
antibody-bound chelating groups followed by TLC analysis
(30). A 50-id aliquot of the labeled antibody solution was
incubated with 25 @il
ofa 0.05M solution of DTPA, pH 6, for
10 mm then the resulting solution was diluted 50-fold with
normal saline and a 3 @d
aliquot was spotted onto a silica gel
TLC plate (Fisher Scientific, Pittsburgh, PA). The plate was
then developed in a 1:1 (v:v) mixture of MeOH and 10%
[email protected] these conditions, antibody-bound ‘
‘
‘In
ac
tivity remained at the origin while unbound activity migrated
closeto the solventfront.
For animal studies, the stock [‘
‘
‘InJB72.3
solution at “.‘l
mCi/mg and 10 mg/ml was diluted with normal saline im
mediately prior to administration to a final concentration of
40 @ig/ml.
NCS
FIGURE1
Structure of the bifunctional chelator used to label B72.3
with 1―ln.
Volume3O •
Number3 •
March 1989
Iodine-l25 Labeling Procedure
Iodine-125 sodium iodide (Atomic Energy ofCanada) (5.0
mCi) was transferredto a 5-ml reaction vial and 100 @lof
0.5M phosphate buffer, pH 7.5, was added. The pH of the
375
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resulting solution was checked and, ifnecessary, readjusted to
7.5. 300 @lof a solution of B72.3 at 1.0 mg/mI in 0. lM
phosphatebuffer,pH 7.5, was then added to the vial, followed
by 50 il offreshly preparedChloramine-i solution at 3.5 mg/
ml in 0.05M phosphate buffer, pH 7.5. The vial was then
immediately capped and the contents mixed for 60 sec before
adding 55 @tl
offreshly prepared sodium metabisulfite solution
at 3.5 mg/ml in 0.05M phosphate buffer, pH 7.5. The vial
wasagain capped and the contents mixed for 30 sec then the
resulting solution was applied to a Sephadex G-50 (Sigma
Chemical Co., St. Louis, MO) column (40 cm). The column
had been pre-treatedto block protein binding sites by eluting
through it 1.5 ml ofa 3% solution ofBSA in 0.O5Mphosphate
buffer,pH 7.5. The ‘251-labeled
antibodywas eluted from the
column with 0.1MTris/0.1SM NaCl, pH 7.5, collecting1-2
ml fractions at 0.5 mi/mm. The desired fractions were pooled
and stored at 2—8°C
until needed. Immediately priorto use in
animal studies,the [‘251]B72.3
preparationwas furtherpurified
by the centrifuged mini-column procedure of Meares et al.
(30), then diluted with normal saline to a final concentration
of 40 @g/ml.The specific activity of the resultingpreparation
was typically 5-10 mCi/mg.
Immunoreactivity Assay
The immunoreactivity of antibody conjugates and radio
labeled antibody preparationswas compared with that of the
underivatized antibody using an enzyme linked immunosor
bent assay(ELISA).As the TAG-72antigenis difficultto
purify in large quantities, the antigen used in this assay was
bovine submaxillary mucin (BSM) (Cooper Biomedical, Mal
vera, PA), an inexpensive and readily available mucin with
which B72.3 is stronglycross-reactive.
Ninety-six well microtiter plates (Dynatech Laboratories,
Arlington, VA) were coated with BSM by incubating in each
well a 100-id aliquot of a 10 @g/mlsolution of BSM in
10 mM Tris, pH 7.4. The plate was covered and incubated
overnight at room temperature then was emptied and washed
twice with deionized water. The plate was then overcoated by
incubating in each well for 2 hr a 0.1% solution of BSA in
PBS (100 id/well). The plate was then stored at 2—8°C
until
needed, with 100 i1 of overcoating solution in each well.
well. The plate was incubated, protected from light, for 15
mm at room temperature then 100 @ilof 0.5M H2S04 was
added to each well. Both the enzyme conjugate solution and
the a-phenylenediamine solution are unstable and these were
prepared no more than 30 mm and 10 mm before use,
respectively. The color generated in each well was read at 490
nm using a microtiter plate reader (Dynatech Laboratories).
Duplicate wells were run at each concentration.
Antibody titration curves were prepared by plotting the
mean optical density at 490 nm against antibody concentra
tion and the curvefor the labeledantibodycomparedwith
that for its underivatized counterpart. A semiquantitative
estimate ofthe immunoreactivity retained after labeling could
be obtained by expressingthe absorbanceof the labeledma
terial as a percentage of the absorbance of the underivatized
antibody at 50% titration. It should be noted that this titration
point, defined in terms of antibody concentration, varies
somewhat depending on the particular lots of BSM and goat
anti-mouse antibody conjugate used in the assay. Thus it is
important that samples of both native and derivatized anti
body are always analyzed at the same time on the same
microtiter plate, in order to provide a valid measurement of
the relativeimmunoreactivity ofthe derivatized material.
Biodistribution Studies in Tumor Bearing Mice
Female athymic mice (nu/nu, BALB/c background)
(Charles River Biotechnology Services, Wilmington, MA)
were injected subcutaneously with S x 106 A375 human
melanoma cells (American Type Culture Collection) in the
left rear flank. 14 days later, 1.25 x l0@LS174T human
colorectal carcinoma cells (American Type Culture Collec
tion) were similarly injected into the right rear flank. After a
further 7-14 days, the solid tumors thatdeveloped had reached
a sizeoffrom 100mg to 500 mg. Animalswerethen injected
intravenously with a mixture of 4 @&g
of [‘251]B72.3
and 4 gig
of [‘
‘
‘In]B72.3
in 200 gil of normal saline, the dose being
administered via the tail vein. At various time points following
the injection, groups of mice (n = 5) were killed and the
tumors plus all internal organs were removed, weighed, and
countedin a gammacounter(PharmaciaLKBBiotechnology
Inc., Gaithersburg,MD). Iodine-125 activity was counted in
a channel with windows set for 15-85 keV and ‘
‘
‘In
activity
Immediatelybefore use, the plate was emptied and washed was countedin a channelhavingwindowsset for 237-257
keV. Cross-countsin the 1251channelwere 5% and in the
five times with PBS.
Parallel ELISA assays were performed on the labeled anti
body and on the underivatized antibody from which it had
been prepared. Both assays were run on the same microtiter
indium channel were 1.5%, gross cpm values being corrected
to compensate for cross-counting. Weighed aliquots of blood,
muscle and skin were counted, as was the residual carcass.
plate, employingserial twofold dilutions of antibody prepa The tail was counted separatelyand animals having >10% of
rations adjusted to an initial concentration of 1—10@g/ml. the dose left in the tail were excluded. A 100-gilaliquot of
Dilutions were made in a solution of 1.0% BSA and 0.1% each labeled antibody preparationwas counted at the same
Tween 20 in PBS. To each well was first added a S0-@laliquot time as the tissues and the activity of each isotope in each
of this dilution buffer followed by a SO-Mlaliquot of the tissue was calculated as a percentage of this injected dose per
antibody solution. The plate was covered and incubated at gram of tissue.
37°Cfor 1 hr then emptied and washed five times with PBS.
Onehundredmicrolitersofa 0.06 @ig/ml
solutionofgoatanti
mouse antibody conjugated to horseradish peroxidase (Kir
kegaard & Perry Laboratories, Gaithersburg@MD) in 0.1%
BSA/0. 1%Tween 20/PBS was added to each well. The plate
was again covered and incubated at 37°Cfor 1 hr, then was
emptied and washedfivetimes with PBS.A 100-idaliquot of
o-phenylenediamine solution prepared from a pre-formulated
kit (Abbott Laboratories, Abbott Park, IL) was added to each
376
Camey,Rogers,andJohnson
RESULTh
Conjugationof Chelator to B72.3
The isothiocyanate derivative of DTPA shown in
Figure 1 reactswith the €-amino
group oflysine residues
TheJournalof NudearMedicine
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TABLE1
to form a thiourea linkage between the chelator and the
antibody. In order to determine the maximum
number
of chelating groups that could be incorporated in this
way into the B72.3 molecule without significant(>20%)
loss of immunoreactivity, a series of conjugation reac
tions were performed at varying input mole ratios of
chelator.antibody. The resulting conjugates were mon
itored for chelator content and immunoreactivity,
typ
ical data from a study ofthis type being shown in Table
Effectof Chelator:AntibodyInputMoleRatioon
SubstitutionLeveland Immunoreactivityof B72.3
ChelatorConjugates
number
ofchelators
per immunoreactivfty
Chalatoranbbody
input (%)1:10.3952:11.0855:13.3747:13.56810:15.04420:115.023
mole ratioAverage conjugateConjugate
1. Although there was some variability from one series
of conjugations to the next, the threshold substitution
level beyond which significant loss of immunoreactivity
was consistently seen was found to be approximately
three DTPA groups per immunoglobulin molecule.
Consequently,
this was the substitution
level chosen for
B72.3-DTPA conjugates used in subsequent animal
studies.
Radiolabeling
of B72.3
important
in that it has been shown that the extent of
tumor uptake of activity is influenced by tumor mass
(32). Mean tumor weightsfor groupsof animalsused
in this study appear in Table 2.
Biodistributions
of 1251and ‘
‘
‘In
labels at 1, 2, 5, and
Indium-i 11 labeling ofB72.3-DTPA conjugates was 7 daysfollowing the injection ofa mixture of['251]B72.3
readily achieved by brief incubation at 10 mg/mi in and [“In]B72.3are shown in Figure 3 and clearance
0.05M citrate buffer, pH 6, with carrier-free [“In] data for key tissues are summarized in Figure 4. There
were no statistically significant differences in levels of
chloride from a commercial source. In order to test the
stability of the B72.3-DTPA conjugate during storage the two labels in any tissue at any time point studied (p
and the reproducibility of radiolabeling with different > 0.1 by two-tailed Student t-test). Nevertheless, whole
lots of' ‘
‘In,
12 labeings were performed on one lot of body retention of activity was consistently somewhat
conjugate over a 10-mo period following its prepara higher for “Inthan for 1251(Fig. 5), these differences
tion. The mean radiochemical yield was 96% (range = being significant (t-test) at 1 day (p < 0.05) and 2 days
93—98%;
s.d. = 1.5%)and there was no loss of labeling (p < 0.001) postinjection but not at the later time
points. No single tissue or organ system was responsible
efficiency over time. Given the consistently high radi
for these differences, but rather there was a general
ochemical yield oflabeled antibody that was obtained,
no attempt was made to develop postlabeling purifica
tion proceduresto remove the small amount of residual
indium activity that was not protein bound.
trend for indium activity in any given tissue to be
slightly higher than iodine activity, as inspection of
sors are superimposable, indicating complete retention
ofimmunological
activity during the respective labeling
creted activity and hence the distribution of activity
between feces and urine is unknown.
Tumor.blood ratio was initially below 1 and rose to
2.6 by Day 7 while the tumor.liver ratio increased from
3.3 on Day 1 to 9.9 on Day 7. The previous observation
Figure 3 reveals. Although these differences never
achieved
Statisticalsignificance in any given tissue, the
Radioiodination
of B72.3 was carried out by the
cumulative
effect over the entire animal was to produce
chloramine-T method. ELISA plots for the [‘25I]B72.3
a
higherwhole-body
retention ofindium than of iodine.
and [“In]B72.3preparations used in the biodistribu
Label
that
was
not
retained
is assumed to have been
tion study appear in Figure 2. In both cases, the curves
excreted,
but
no
attempt
was
made to quantitate cx
for the labeled antibody and its underivatized precur
processes.
Biodistribution of f'@I]B723 and f―InIB72.3
Coadministered to Athymic Mice Bearing Human
Colon CarcinomaXenografts
The animal model used in this study has been de
scribed previously
by Colcher
and co-workers
(8,21,28,31). LS174T is a human colon carcinoma line
that expressesthe TAG-72 antigen recognizedby B72.3,
while A375 is a human melanoma which does not
that B72.3 exhibits prolonged binding at the tumor (28)
was confirmed,in that LS174Tlevelsof both isotopes
remained essentially constant throughout the course of
the study. That this was an antigen dependent phenom
enon was evident from the ratio ofLSi74T:A375 activ
ity, which rose from 3.3 on Day 1 to 7.5 on Day 7.
express TAG-72 and serves as a control for nonspecific
uptake ofimmunoglobulin
into such xenografts. As the
DISCUSSION
colon carcinoma grows more rapidly, the melanoma
was implanted earlier so that tumor weights would fall
within the same range at the time of study. This is
To the best of our knowledge, this is the first study
to have demonstrated identical biodistributions for 1251
and “In-labeled
preparationsofthe same antibody.
Volume30 •
Number3 •
March 1989
377
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2.5-
A
2.0A490
1.5-
1.0-
05-
N
0-
@
I
10
I
i
I
5
2.5 1.25 0.625
AntibodyConcentration (,@gIml)
ao -
0
B
2.@-
2.0-
15-
1.0—
FiGURE 2
ELISA plots for
(-•
[@lnJB72.3
•) and the underivatized
B72.3 from which it was prepared
(-O--—O-) (panel A) and for [1251]
@
05
B72.3 F• •-)and the
atized B72.3 from wh@hit was pre
0
h@
‘@
05
pared(-O---—O-)(panelB).
0.25
0.125
0.062
o.d15
Antibody Concentration
An analogous dual isotope study using the same
antigen/antibody
system and mouse model employed
in this work was recently reported by Brown et al. (33).
Selected data from that study are compared with those
0
(ag/mI)
two studies may also have played a part in producing
this disparity. Certainly, the absolute LS174T uptakes
reported by Brown et al. (32 %ID/g for 1251and 42
%ID/g for ‘
‘
‘In)are higher than those reported by
from this work in Table 3. The 1251levels found in
others (8,21,27,28) and this, combined with reduced
blood and all normal tissues are strikingly similar be
tween the two studies, suggesting that minor differences
blood levels of “Indue to higher nontarget clearance,
produced tumor.blood ratios that were substantially
in methodology and labeling technique (e.g., Brown et
al. employed the Iodogen method to radioiodinate
B72.3) do not produce widely disparate results in this
model.
The only tissue to show a graphic difference in 1251
uptake between the two studies is the LS174T tumor.
This probably reflects the inherent variability of such
xenografts
that is apparent
from the large standard
deviations for activity levels in the tumor compared to
all other tissues. Differences in mean tumor weights
and/or the particular passage of LS174T used in the
378
Camey,Rogers,andJohnson
TABLE 2
MeanTumorWeights(n =5)
Dayof
MeanLS174T
MeanA375
kill time
weight (g (±s.d.))
weight (g (±s.d.))
10.36(0.11)0.40(0.48)20.66(0.29)0.24(0.17)50.44
(0.10)0.39(0.35)70.36
(0.09)0.21
(0.08)
TheJournalof NuclearMedicine
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A
20
B
20
18
18
U)
Cl)
Cl)
Cl)
@@::
.1z
El4
E14
(@
L@
@12
@12
&@@)10
@-
Q@10
Cl)
Cl)
0
0
@8
•0
•0
t6
G)
@E'
t6
a)
_4
0
@
0
2
0
2
0
BloodLiverKidneyLS174T
A375SpleenLungs
C
20
BloodLiverKidneyLS174T
A375SpleenLungs
D
20
18
18
G)
0)
(1)16
@16
Cl)'
l@
I::
E14
C@
‘-
@12
@12
@-
‘@10
Cl)
U)
0
0
•D
0)
@0
0)
08
t6
0)
@E
0
0)
@4
0
4
2
2
0
0
BloodLiverKidneyLS174T
A375SpleenLungs
FIGURE3
BloodLiverKidneyLS174T
P375SpleenLungs
Biodistnbutionin athymicmice bearingLSI 74T xenograftsof 1251
(U) and
@ln
(D) activityat 1 day (panelA), 2 days
(panelB),5 days(panelC) and7 days(panelD)followingi.v. injectionof a mixtureof i251]B72.3and [“1In]B72.3.
higher in the Brown Ctal. study (-@-4
for ‘
‘
‘In
and 2 for
1251) than
in
the
present
study
(‘@-i for
both
labels).
When normal tissue levels of “Inseen in the two
studies are compared (Table 3), it is apparent that non-
Volume30 •
Number3 •
March 1989
targetuptake ofthis label is substantiallylower, and the
blood
level
correspondingly
higher,
in
the
present
study.
The co-administration study ofBrown et al. shows over
sixfold more ‘
‘
‘In
in the liver than 125!,threefold more
379
Downloaded from jnm.snmjournals.org by on April 30, 2014. For personal use only.
20
a)
0
18
LS174T
16
a)
C
C
E
U)
U)
a)
I— 14
a)
0
E
Co
C')
0
12
•0
G)
0
a)
C
10
8
Day 1
Day 2
Day 5
Day 7
FIGURE5
I
Blood
6
Whole-body retention of 1251(D) and
@In
4 activity follow
ingi.v.injectionof a mixtureof ‘25I-B72.3
(45giCi)and
@In
(4 giCi)into athymic mice bearing LS174T xenografts.
4@
2
Liver
0
1
2
3
4
5
6
7
Time (Days)
FIGURE4
@
Clearance curves for 1@In(—) and 1251(@ @)
activity
followingi.v. injectionof a mixtureof [‘25l]B72.3
and[@In]
B72.3 into athymic mice bearing LS174T xenografts.
moiety in (A) to regenerate two carboxymethyl groups
and give the DTTA conjugate (B) (Fig. 6). However,
should the anhydride moiety of(A) encounter an amino
group on a neighboring antibody molecule prior to
hydrolysis, the cross-linked species (C) results. Such
intermolecular cross-linking has long been recognized
as a potential problem in the bicyclic DTPA anhydride
conjugation ofproteins (34,35) and reaction conditions
are usually chosen such as to minimize the formation
the kidney than 1251,while blood ‘
‘
‘In
activity is only
of such species, although it is generally not possible to
completely eliminate them. The formation of intermo
lecularcross-linksis readilydetected by techniques such
as size exclusion chromatography or gel electrophoresis
and is sometimes apparent from immunoreactivity as
75% that of the 125!level. These data cannot be cx
says if the cross-link disrupts an antigen binding site.
planed on the basis of impaired immunoreactivity, as
The third possible fate of (A) is for the anhydride
moiety to react with a second amino group present in
the same immunoglobulin molecule, producing an in
tramolecularcross-link (D) (Fig. 6). If such a cross-link
11‘In in
the
spleen
than
1251,
and
fourfold
more
‘
‘
‘In in
the [‘
‘
‘In]B72.3preparation used by Brown et al. was
reported to be fully immunoreactive by competitive
solid phase RIA (33). The most obvious
difference
between the present work and that of Brown et al. is
that the latter study employed the bicyclic DTPA an
hydride procedure (18) to prepare [“InJB72.3.An
independent study in this same model using [“In]
does not involve lysine residues in or near an antigen
B72.3 produced by the bicyclic anhydride method (21)
from first principles, it might be expected that intra
showed liver activity at 48 hr postinjection of 17.20
of 17. 14 %ID/g, and blood activity of 10.67 %ID/g,
molecular cross-linking would occur much more fre
quently than intermolecular cross-linking. The anhy
dride moiety in (A) is tethered in close proximity to the
indicating that this biodistribution pattern is reasonably
reproducible between different laboratories.
concentration
%ID/g, spleen activity of 9. 14 %ID/g, kidney activity
When the bicyclic anhydride ofDTPA reactswith an
binding site, thus producing no loss of immunoreactiv
ity, it is undetectable by any of the methods routinely
used to evaluate
antibody-chelator
conjugates.
Yet,
first antibody molecule whereas, even at an antibody
of 10 mg/mi, there is an average of only
one IgG molecule per 108A3, meaning that the second
amino group present on an antibody, the intermediate
(A) (Fig. 6) is transiently formed. There are three pos
sible fates for this intermediate. The desired reaction
dride moiety than are other lysine residues present in
pathway involves hydrolysis ofthe remaining anhydride
the same molecule. It is our hypothesis that species of
380
Carney,
Rogers,andJohnson
antibody molecule needed to make an intermolecular
cross-link is substantially more distant from the anhy
The Journalof NuclearMedicine
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3
Biodistnbution Data frTABLE
om Co-Administration
L5174T%
Studiesof [125lJB72.3
and [@In]B72.3 in Athymic
Colon Carcinoma XenograftsMice
Bearing
postinjection1@I
Injecteddose/g (mean(±s.d.),
n = 5) at 48 hr
“In111lnOrgan(Ref.
1251
work)Blood16.26
33)
(thiswork)
(5.51)16.38(1.18)Spleen3.86(7.46)
16.60(1.22)
(1.12)
(0.25)Uver3.07
(0.48)Kidney3.18
(1.19)
(1.02)
(0.15)Muscle1
(0.41)1.66(0.27)Heart3.50
.09 (0.40)
.30)6.27(0.80)Lung7.35
(1.63)
(0.79)01
(3.06)
tract1
(0.39)1.73(0.17)LS174T32.50
.06 (0.29)
(10.26)
(Ref.33)(this
11.23
3.42(0.27)
12.12 (7.25)3.91
3.62(0.51)
3.34(0.14)
1.61(0.29)
6.15 (0.79)
19.39(10.97)4.12
12.64 (1.66)3.51
1.00
2.95 (1
6.38(0.81)
1.36(0.15)
17.54 (3.65)
6.15 (2.52)6.41
2.03
42.22 (15.14)17.96(3.54)
cross-links that do
as a second major uncertainty is inherent in the bicyclic
not affect immunoreactivity, are recognized as foreign
DTPA anhydride approach. The desired species (B)
contains the seven-coordinate DTTA residue and there
type (D), containing
intramolecular
by the reticuloendotheial
system and that this is a
major cause of the observed liver uptake of―‘In
from
has been some debate as to whether this chelator pos
bicyclic DTPA anhydride conjugates.
sesses sufficient stability to completely
It is difficult to be more absolute in this argument,
+
@
NN:
:@-‘,N
retain ‘
‘
‘Inin
vivo (31,34,36). Species (C) and (D) (Fig. 6), on the
_____________
L
+H@O
,—CO,H
(B)
(D)
HO@@
. NH,
(C)
. NH,
FIGURE6
Schematicrepresentationof productsfrom reactionof the bicyclicanhydrideof DTPAwith an antibody.
Volume3O •
Number3 •
March 1989
381
Downloaded from jnm.snmjournals.org by on April 30, 2014. For personal use only.
other hand, contain a residual six-coordinate metal
binding site comprising the three amine nitrogens of
the diethylenetriamine framework and the three re
maiming carboxylic acid groups. Little is known about
the stability ofchelate complexes formed by such a six
coordinate system, but it may be safely assumed that
the formation constants for such complexes do not
exceed those of the six-coordinate chelator EDTA, and
may well be substantially lower. Steric constraints aris
ing from both ends ofthe chelating moiety being bound
to bulky immunoglobulin backbones may make opti
mum folding of this potentially six-coordinate residue
a very unfavorable process. Thus, in addition to protein
denaturation
phenomena,
the formation
of cross-links
also produces a population of immunoglobulin mole
identical rates for cleavage of the iodine-tyrosine
bond
and of the thiourea linkage, and this seems unlikely.
The more probable scenario is that the 1251label re
maimedbound to tyrosine and the ‘
‘
‘In
label remained
bound to lysine throughout the course ofthe study and
that catabolic processes produced peptides (or even
single amino
acids) containing
these bound
labels,
metal binding properties. It is therefore entirely possible
which were then cleared with the label still in place.
The data presented here are in basic agreement with
prior reports (21,31) that, in this model, B72.3 conju
that instability of some, if not all, of these chelation
gates prepared
sites may contribute
ofDTPA show reducedliver, spleen, and kidney uptake
cules with poorly defined, and probably quite variable,
‘
I ‘In-labeled
to the high liver activity seen with
antibodies
prepared
via
the
bicyclic
anhy
dride of DTPA, as typical preparations probably con
using benzylisothiocyanate
derivatives
compared to analogous conjugates obtained by the
bicycic anhydride method. Our findings and those of
tamallthree
species
(B-D).
these prior studiesare, however,divergentin a number
While the bicycic anhydride approach to covalent
linkage of DTPA to proteins offers the worst case of a
ofrespects. The reduced normal tissue uptakes reported
by Brechbiel et al. (31) and Esteban et al. (21) were
achieved only after extensive postlabeling purification
ofthe [‘
‘
‘In]B72.3
preparationsusing a combination of
stoichiometric
cross-linking agent (one potential cross
linking moiety per activated DTPA molecule) other
approaches to the selective derivatization of one of the
five carboxymethyl groups, whether through a carbox
ycarbonic anhydride (1 7) or through an active ester
(19), suffer many of the same disadvantages. Control
of reaction stoichiometry so as to completely avoid the
formation
of di-activated
species is very difficult (37)
Sephadex G-50 gel permeation chromatography
and
size exclusion HPLC, whereas the data presented here
were obtained without benefit of postlabeling purifica
tion. Undoubtedly, the lower nontarget uptakes seen in
the Esteban et al. and Brechbiel Ct al. studies reflect
their use of a noncross linking DTPA derivative that
and any such species formed are capable of producing
retains the complete donor atom array of the parent
cross-links. Although, unlike the bicycic anhydride,
these species constitute only a small percentage of the
molecule. Their data do, however, illustrate that addi
tional factors, such as the number of chelating sites
activated DTPA preparation, if intramolecular cross
introduced
linking occurs more frequently than intermolecular
cross-linking and if the former causes elevated reticu
loendothelial uptake, then the presence of even small
of the preparation with respect to adventitious metal
ion contamination, must also be taken into account
and controlled. It is factors such as the latter that
probably account for the observation that liver and
amounts of di-activated species could skew the biodis
tribution patterns subsequently seen, given that the
activated DTPA preparation is usually added to the
antibody in large excess.
@
case would arise if catabolism of the chelate conjugate
were to result, not in the release of free indium, but in
cleavage of the thiourea bond and release of chelated
indium. The latter would be expected to clear rapidly
through the urine, as does free iodide and this, in
principle, could result in parallel distributions of the
two labels. However, it would be necessary to invoke
into each antibody molecule and the purity
kidney activity at 48 hr postinjection were twofold and
threefold higher, respectively, in the Esteban et al. study
than in our own (21). These same factorsare also likely
When ‘
‘
‘In
labeling is accomplished by procedures to be responsible for the most significant disparity be
that do not produce protein cross-linking and retain all tween these two studies, namely that a [‘251]B72.3/
co-administration
study
performed
by
eight metal binding sites of the DTPA molecule, the [I ‘In]B72.3
data in Figures3 and 4 suggestthat ‘
‘
‘In
can becomea
Esteban et al. failed to demonstrate
legitimate tracer of the biologic fate of immunoglobu
lins. The parallel biodistributions of 1251and ‘
‘
‘In
seen
in this study are best explained by assuming that they
butions of the two labels. It is reported that, although
reflect the physiological processes normally involved in
tinued to rise whereas those for ‘
‘
‘In
did not. Thus, for
example, tumor.liver ratio was 5 at 7 days postinjection
for [“In]B72.3as opposed to 14 for [‘251]B72.3.
Such
immunoglobulin
uptake and catabolism. Ifthe 1251label
were being cleaved from tyrosine and/or the ‘
‘
‘In
label
being released from the chelate during catabolism,
markedly different distributions ofthe two labels would
be expected to result at the later time points. A special
382
Camey,Rogers,andJohnson
parallel biodistri
tumor.organ ratios were very similar for both labels for
the first 3 days, thereafter 1251tumor:organ ratios con
results serve to emphasize that any one of a number of
methodological problems can have the effect of produc
ing disparate 1251and ‘
‘
‘Indistributions. Only truly
TheJournalof NuclearMedicine
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parallel behavior of the two labels permits a degree of
confidence that the intrinsic behavior of the antibody
molecule is being observed unobscured by label-related
artifacts.
method.Science 1983;220:613—615.
REFERENCES
1. Larson SM. Radiolabeled monoclonal anti-tumor an
tibodies in diagnosis and therapy. J Nuci Med 1985;
26:528—545.
2. Goldenberg
ticarcinoembryonic
antigen monoclonal antibody
ZCE-025.Radiology1987;164:617—621.
17. Krejcarek GE, Tucker KL. Covalent attachment of
chelatinggroups to macromolecules. Biochem Biophys
Res Commun 1977; 77:58 1—585.
18. Hnatowich DJ, Layne WW, Childs RL, et al. Radio
active labeling of antibody: a simple and efficient
DM.
Current
status
of cancer
imaging
with radiolabeled antibodies. J Cancer Res C/in Onco!
1987;113:203—208.
3. Begent RHJ. Recent advances in tumor imaging: use
of radiolabeled antitumour antibodies. Biochim Bio
19. Paxton RI, Jakowatz JG, Beatty JD, et al. High spe
cific activity ‘
‘
‘In-labeledanticarcinoembryonic anti
gen antibody:improved method for the synthesisof
diethylenetriaminepentaacetic acid conjugates Cancer
Res 1985;45:5694—5699.
20. Meares CF, Goodwin DA. Linking radiometals to
proteins with bifunctional chelating agents. J Protein
Chem 1984;3:215—228.
21. Esteban JM, Schiom J, Gansow OA, Ct al. New
method for the chelation of indium-l 11 to mono
clonal antibodies: biodistribution and imaging of
athymicmice bearinghuman colon carcinomaxeno
grafts.JNuclMed 1987;28:861—870.
physActa1985;780:151—166.
4. Goldenberg DM, Preston DF, Primus SJ, et al. Pho
toscan localization of GW-39 tumors in hamsters us
inganticarcinoembryonicantigenimmunoglobulinG.
CancerRes1974;34:1—9.
5. Mach JP, Carrel S, Merenda C, et al. In vivo localiza
22.
Rainsbury
RM, Ott RJ, Westwood
JH, et al. Location
of metastatic breast carcinoma by a monoclonal anti
body chelate labeled with indium-l 11. Lancet 1983;
tion of radiolabeledantibodies to carcinoembryonic
ii:934—938.
antigenin human colon carcinomagraftedinto nude
23. Halpern SE, Dillman RO, Witztum KY, et al. Radio
mice. Nature 1974; 248:704—706.
immunodetection of melanoma utilizing In-i 11 96.5
6. Beaumier PL, Krohn KA, Carrasquillo JA, et al. Mel
anoma localization in nude mice with monoclonal
monoclonal antibody: a preliminary report. Radiology
1985; 155:493—499.
Fab against
p97.JNuclMed 1985;26:1172—1179.
7. ShahSA, GallagherBM, SandsH. Radioimmunode 24. Epenetos AA, Snook D, Hooker G, et al. Indium-l 11
tection ofsmall human xenografts in spleen of athymic
mice by monoclonal antibodies. Cancer Res 1985;
45:5824—5829.
8. Keenan AM, Colcher D, Larson SM, et al. Radioim
munoscintigraphy of human colon cancer xenografts
in mice with radioiodinated antibody B72.3. J Nuci
Med 1984;25:1197—1203.
labeled monoclonal antibody to placental alkaline
phosphatase in the detection of neoplasms of testis,
ovary and cervix. Lancet 1985; ii:350—353.
25. Johnson DK, Westerberg DA, Carney PL, Ct al. Prep
aration and biodistribution of indium-i 11 labelled
chelate immunoconjugates of a monoclonal antibody
to carcinoembryonic antigen. Br J Cancer 1987;
@6:5
13—5
14.
9. Larson SM, Brown JP, Wright PW, et al. Imaging of
melanomawith 1-131labeledmonoclonalantibodies@ 26. Nuti M, Teramoto YA, Mariani-Costantii R, et al.
A monoclonal antibody (B72.3) defines patterns of
distribution of a novel tumor-associated antigen in
JNuclMed 1983;24:123—129.
10. MoldofskyPJ, Powe J, MuihernCB, ci al. Metastatic
colon carcinoma detected with radiolabeled F(ab')@
monoclonal antibody fragments. Radiology 1983;
149:549—555.
11. Pateisky N, Philipp K, Skodler WD, et al. Radioim
munodetection in patients with suspected ovarian can
cer.
JNuclMed 1985;26:1369—1376.
12. Delaloye B, Bischof-Delaloye A, Buchegger F, Ct al.
Detection of colorectal carcinoma by emission-com
humanmammarycarcinomacellpopulations.mt j
Cancer 1982;29:539—545.
27. Lundy J, Mornex F, Keenan AM, et al. Radioimmu
nodetection of human colon carcinoma xenografts in
visceral organs of congenitally athymic mice. Cancer
1986;57:503—509.
28. Colcher D, Keenan AM, Larson SM, et a!. Prolonged
binding of a radiolabeled monoclonal antibody
puterized tomography after injection of ‘23I-labeled
Fab or F(ab') fragmentsfrom monoclonal anti-car
(B72.3) used for the in situ radioimmunodetection of
humancolon carcinomaxenografts.CancerRes 1984;
cinoembryonic antigen antibodies. J Clin Invest 1986;
77:301—311.
13. Rainsbury RM, Westwood JH. Tumour localization
44:5744-5751.
29. Thor A, Ohuchi N, Szpak CA, et al. Distribution of
oncofetal antigen tumor-associated glycoprotein-72
with monoclonal antibody radioactively labelled with
defined by monoclonal antibody B72.3. CancerRes
metal chelate ratherthan iodine. Lancet 1982; ii:l347-
1348.
14. Hnatowich DJ, Griffin 1W, KosciuczykC, et al. Phar
macokinetics of an indium-i 11 labeled monoclonal
1986;46:3118—3
124.
30. Meares CF, McCall MJ, Reardan DT, et al. Conjuga
tion of antibodies with bifunctional chelating agents:
isothiocyanate and bromoacetamide reagents, meth
antibody in cancer patients. JNuclMed 1985; 26:849858.
15. Beatty JD, Duds RB, Williams LE, et al. Preoperative
3 1.
imaging of colorectal carcinoma with “In-labeled
anticarcinoembryonic antigen monoclonal antibody.
Cancer Res 1986; 46:6494—6502.
16. Abdel-Nabi HH, Schwartz AN, Higano CS, Ct al. 32.
Colorectal carcinoma: detection with indium-i 11 an
Volume30•
Number3•
March1989
ods ofanalysis, and subsequent addition ofmetal ions.
AnalBiochem
1984;142:68—78.
Brechbiel MW, Gansow OA, Atcher RW, et al. Syn
thesis of l-(p-isothiocyanatObenzyl) derivatives of
DTPA and EDTA. Antibody labeling and tumor
imagingstudies.
morgan Chem 1986;25:2772—2781.
Williams LE, Duda RB, Proffitt RT, et al. Tumor
uptake as a function of tumor mass: a mathematical
383
Downloaded from jnm.snmjournals.org by on April 30, 2014. For personal use only.
model. JNuclMed
33.
Brown
BA, Comeau
1988; 29: 103—109.
RD,
Jones
PL, et al. Pharmaco
kinetics of the monoclonal antibody B72.3 and its
fragments labeled with either 1251or ‘
‘
‘In.Cancer Res
1987;47:1149—1154.
34. Hnatowich DJ, Childs RL, Lanteigne D, et al. The
preparation ofDTPA-coupled antibodies radiolabeled
with metallic radionucides: an improved method. J
ImmunolMeth 1983;65:147—157.
35. Paik CH, Ebbert MA, Murphy PR, et al. Factors
384
Camey,Rogers,andJohnson
influencing DTPA conjugation with antibodies by
cyclic DTPA anhydride. J Nuci Med 1983; 24:1158—
1163.
36. Hnatowich DJ. Biodistribution ofindium-1 11 labeled
monoclonal antibodies. J Nuci Med 1987; 28:1924—
1925.
37. Paik CH, Murphy PR, Eckelman WC, et al. Optimi
zation of the DTPA mixed anhydride reaction with
antibodies at low concentration. J Nucl Med 1983;
24:932—936.
The Journalof NuclearMedicine