[CANCER RESEARCH (SUPPL.) 50. 780s-782s. February 1. 1990]
Development of Radiochemically Pure Antibodies1
W. C. Eckelman
Squibb Institute for Medical Research, Aviv Brunswick, New Jersey 08903-0191
Abstract
There are presently three approaches to radiolabeling antibodies:
direct radiolabeling; radiolabeling of a chelating agent conjugated to an
antibody; and radiolabeling of a chelating agent before conjugation to an
antibody. Using either the direct radiolabeling or radiolabeling of a
chelating agent conjugated to an antibody has not led to a radiochemically
pure ""Tc-labeled protein. High radiochemical purity is obtained using
a prelabeled ligand; therefore this method is preferred.
One of the major efforts since the first Conference on Radioimmunodetection and Radioimmunotherapy
of Cancer has
been the development of antibodies that are radiochemically
pure in vitro and remain so in vivo. Since radioiodine has been
one of the first radionuclides used to label antibodies, many
studies have centered around the preparation of radiochemically
pure antibodies either by direct iodination or by indirect labeling
through a reagent such as the Bolton-Hunter ligand. The cur
rent research emphasis is on in vivo stability using various
iodinated species less susceptible to enzymatic deiodination (1,
2).
There have been two approaches to radiolabeling antibodies
with metallic radionuclides using bifunctional chelates; either
the chelating agent is first attached to the antibody followed by
radiolabeling or the chelating agent is first radiolabeled and the
resulting chelate is bound to the antibody. Very few experiments
have shown that first attaching the chelating agent to the
antibody results in an "instant" kit in which all the radiolabel
is bound via the chelating agent. For 99mTc,the indirect labeling
methods have mostly involved the use of DTPA- bound to the
protein. DTPA is considered a bifunctional chelate in that it
binds to the protein and to the "Tc. Other chelating agents,
derivatives of DADS (3), derivatives of bis-A'-methylthiosemicarbazone (4), and metallothionein (5), have been conjugated
to antibodies for "Tc labeling. Although 99mTclabeling con
ditions and biodistribution studies of the labeled products were
reported, a systematic investigation of the effectiveness of this
latter chelator for complexing "Tc in competition with anti
body has been reported only in abstract form (6).
Hnatowich's group (7, 8) has carried out various experiments
to determine the expected ratio of "Tc binding to the chelating
agent in the bifunctional chelate compared to direct binding to
the antibody. Using 50 Mg/ml DTPA as the molar equivalent
to 20 mg/ml IgG, (0.13 ^mol/ml) various binding conditions
were tested. Without protein present, DTPA complexed re
duced "Tc efficiently, but not quantitatively (Table 1).
In a subsequent paper, they determined the appropriate
tin:DTPA ratio. As has been observed earlier, too little stannous
ion resulted in a high percentage of pertechnetate and too much
stannous ion resulted in tin colloid. The labeling of conjugated
protein via the bifunctional chelate is about 80%. The distri
bution in normal mice at l h is similar for '"In-DTPA-IgG and
"Tc-DTPA-IgG.
Three N2S2 (diaminodithio) ligands were studied by Liang et
' Presented at the "Second Conference on Radioimmunodetection and Ra
dioimmunotherapy of Cancer." September 8-10. 1988, Princeton, NJ.
2The abbreviations used are: DTPA. diethylenetriaminepentaacetic
acid;
DADS. bis(mercaptoacetyl)ethylenediamine; DADT. diaminodithiol.
al. (9) to determine if they had advantages over DTPA as
bifunctional chelates of antibodies (Table 2). Optimal concen
trations of tin and chelator were determined to be 2.5 to 5 /ig/
ml SnCl2-2H2O and 50 Mg/ml of chelator. The optimal pH
range was 5 to 8. Labeling in the presence of 5 mg/ml of IgG
was used to determine the percentage expected to be labeled via
the N2S2 chelating agent and the percentage expected to be
directly bound to the antibody (Table 3).
The DADT-3C-2OH in which the ethylene backbone is re
placed with a propylene backbone has the highest percentage
binding. This appears to be the only example of a chelating
agent that can compete quantitatively with an equimolar con
centration of IgG.
If the source of 99mTcis 99mTc-EDTA at 100 Mg/ml, then the
requirement for chelator concentration is less (Table 4). Using
150,000 as the molecular weight of IgG there are 33 nmol/ml
of IgG. If the molecular weight of the free amine is used (236),
then 3 ng of DADT are 13 nmol/ml and 8 Mgof DADT are 34
nmol/ml. These molar values do not agree with the molar ratios
stated by Liang et al. (9). However, it appears that the chelation
is more effective when 9VrnTc-EDTA is used than when the
chelator is labeled directly.
Yokoyama's group (10) has developed the neutral 99mTc
chelate ketobisthiosemicarbazone
for use in bifunctional che
lates. Various derivatives have been prepared including the pcarbonylethylphenylglycol
derivative, which has been conju
gated to antibody by activating the acid group using the phosphorylazide method. Stannous ascorbate was used as the reduc
ing agent and 99mTc-ascorbate as the transfer agent. As in most
of these studies the optimal pH is between 4.5 and 6.2. The
labeling experiments were carried out to determine what per
centage of the 99mTc binds to the chelating agent and what
percentage binds directly to the antibody. At ratios of chelating
agent to antibody that preserve the immunological activity (i.e.,
1:1 molar ratio), about 15-20% of the "Tc is directly bound
to the protein. In vitro and in vivo stability of the derivatized
antibody is high. Although direct binding of 99mTcto antibodies
has been reported (11-14) the stability and inertness of the
bond are in question. There appear to be two binding sites on
the antibody, one with high affinity and one with lower affinity.
Steigman et al. (15) were the first to suggest that sulfhydryl
groups were responsible for the direct binding of 99mTc.Paik et
al. (16, 17) substantiated this hypothesis by carrying out a
number of competitive experiments and by titrating the sulfhy
dryl groups using Ellman's reagent. The available sulfhydryl
groups were determined after 30 min incubation of stannous
chloride with antibody. The stannous chloride was present in
10-fold excess over the protein. The results obtained are re
ported in Table 5.
The presence of both high affinity sites and low affinity sites
on the protein was defined by competition studies using DTPA.
The high affinity sites were determined as those which bound
99nTc even at large molar excesses of DTPA. The percentage
bound to protein was determined at various ratios of DTPA to
protein.
These results are obtained only under conditions of direct
competition between DTPA and antibody. If the protein is
labeled first, then DTPA is effective only at high pH. One
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RADIOCHEMICALLV
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Table 6 Percentage of loss of radioactivity from n"Tc-labeled antibodies"
Table l DTPA binding ofstannous reduced """Tc"
bound40
bound81
DTPA (fig/ml)25
Challenge1
50
6
8991pH7
100pH6
6%
' Taken from Lanteigne and Hnatowich (7).
DTPA1
mM
mM N2S2
6 M ureaDirect
" From Fritzberg (20).
59
80
77%
Table 2 Various diaminodilhiot derivatives"
Table 3 Labeling o/jVA compound in the presence of 5 mg/ml IgG"
chelateDADT-HM*
"Te
DADT-3C-OH42
98
94
98
95
96
78
89
92
96
10:1DADT8
58%
°Taken from Liang et al. (9).
* DADT-HM. hexamethyl DADT.
covalently bound chelating agent, do .not achieve high radiochemical purity, investigators have turned to prelabeling the
ligand and then reacting this ""Te chelate with the antibody.
Table 4 Labeling of DADT by trancomplexation from v""Tc-EDTA in the
presence of 5 mg/ml lg(i
DADT0(^g/ml)3
(molarratio)2
8DADT:IgG
' As stated by Liang et al. (9).
5%
"Te
Fritzberg et al. (3, 18) have prelabeled the DADS ligand and
then bound the ligand to the antibody via an activated ester.
Likewise Franz et al. (19) have prelabeled the /V-propylamine
derivative of cyclam with 99mTcand then reacted that chelate
with an activated antibody. This method assures that the 99mTc
asTc-DADT85
88
is bound to the chelating agent and, therefore, that a single
radiochemical should be produced. On the other hand, the
development of an "instant kit" will be difficult with this
Table 5 Sulfhydryl groups present for antibody type
Antibody
IgG
F(ab')2
Fab
" Mean ±SD.
0
1.9
of Ab activity
recovered''
no. of MAC ligands/
Ab
molecule2.8
(mean
SD)89.5
±
±0.2'
±1.0
84.2 ±2.1
±0.24.2
3.1
30
89.7 ±2.3
±0.5
100150Av.
88.3 ±1.8
3.6 ±0.3%
"MAC, aminopropyl cyclam: MBS. m-maleimidobenzoyl-A'-hydroxysuccinimide; Ab. antibody: SH, sulfhydryl.
* The average number of MAC ligands conjugated to each rabbit anti-human
serum albumin-lgG molecule (Ab) was estimated assuming ""Tc-MAC-SH had
equal reactivities with MBS-Abs.
' MBS-Abs were prepared by incubating Abs in different molar ratios with
MBS. From Franz et al. (19).
d The percentage of Ab reactivity recovered was determined by affinity chromatography and represents the percentage of Ab binding of MAC Abs compared
to unconjugated Abs.
' Mean ±SD.
' Taken from Liang et al. (9).
61
8255
54.0Bifunctional 32.0Prelabeled
ligand0
MBS:Ab
rat¡or10
I
OH
2:1
3:15:1
chelate0.7
Table 7 Average number of MAC" ligands" conjugated per antibody molecule at
different MBS:antibody ratios'
HSCH2CH2NHR,NHCH2CH2SH
DADT = R, = CH2CH2
DADT-HM = R, = CH2C(Me)2
DADT-3C-20H = R, = CH2CHCH2
Compound:IgG
(molarratio)1:1
labeled0.5
Sulfhydryl groups/antibody
5.5 ±0.7"
4.2 ±0.6
0.9 ±0.15
hypothesis is that the high affinity binding to IgG involves a
Tc(V) species whereas the binding to DTPA involves a Tc(III)
or Tc(IV) species. Competitive experiments indicate that the
distribution of 99mTcbetween the competing ligands is deter
approach. Fritzberg (18, 20) has compared the use of direct
labeling a bifunctional chelate and a preformed chelate to
determine relative radiochemical purity and stability. He used
the 2,5-dimercaptoacetamidopropanoate
as the ligand. The core
ligand (Tc-DADS) has been suggested as a 99mTcsubstitute for
hippuran. In the case of bifunctional chelate, the ligand was
conjugated to antibody using an activated ester. The antibodyligand complex was then radiolabeled by ligand exchange. For
mined by the forward rate constants but not by the equilibrium
the prelabeled ligand, the diamidodithio ligand was radiolabeled
constants for these reaction conditions. This direct labeling
first, the acid was activated to the ester, and then the prelabeled
approach has been refined by others to maximize the percentage
ligand was reacted with the antibody. The relative stabilities
of the free sulfhydryl groups. Paik et al. estimate that about 10 were tested by incubation of each radiolabeled antibody type
times molar excess of DTPA is required to prevent 99mTc with either DTPA or excess diamidodithio ligand (Table 6).
complexation with low affinity sites. However, the presence of
It is clear from this experiment that the prelabeled ligand
free DTPA reduces drastically the percentage of 99mTcbound
forms the most stable bond. Esterification of the preformed
to antibody.
technetium complex went in about 70% yield. Conjugation
yields using 2.5 mg/ml F(ab')2 at pH 9 were between 50 and
One possible solution for this low yield is to reequilibrate the
mixture of low and high affinity sites in the presence of DTPA
70%.
at high pH. This apparently increases the percentage of tech
Franz et al. (19) have used a derivative of the well character
netium on the high affinity sites. Distribution studies in mice
ized cyclam to avoid direct antibody binding. Cyclam was
derivatized at the 1-nitrogen with a 3-aminopropyl derivative.
show similarities among the antibodies labeled by different
The cyclam derivative was treated with 2-iminothiolate to form
techniques at the high affinity sites (16, 17). The generally
the imidate. This was reacted with activated IgG to form the
lower % D/g values for the antibody containing both strongly
and weakly bound 99mTcindicates that a soluble chelate rather
bifunctional chelate. The relationship of activated antibody and
than pertechnetate may be the major radiochemical impurity.
number of chelates incorporated are given in Table 7.
Because both the direct labeling methods, which apparently
These experiments show one of the problems with using
depend on free sulfhydryl groups for binding 99mTc,and the
prelabeled ligands. Only 4% of the ""Te becomes bound to the
bifunctional chelate approach, which depends on a competition
between weak and strong direct binding and binding by the
IgG. A more efficient coupling reaction is needed in this case.
If the ligand is labeled with 99mTcin the presence of the activated
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RADIOCHEMICALLY
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coupling group, then optimal radiolabeling conditions may not
be available. On the other hand, if the technetium complex is
activated toward conjugation after formation, another proce
dural step is required. This balance must be dealt with to obtain
maximum efficiency.
The ideal nuclear properties of "Tc warrant further defini
tion of the chemistry of technetium. To date, the labeling of
antibodies with 99mTcusing an "instant kit" has not resulted in
a product with high radiochemical purity. High radiochemical
purity is obtained using the prelabeled ligands, but this neces
sitates a multistep kit. However, in light of the large number of
variables involved in each experiment with radiolabeled mono
clonal antibodies, it seems prudent to use only preformed
ligands, not only with "Tc, but with all metallic radionuclides,
until such time that an ideal combination of radionuclide and
antibody can be identified. The use of directly labeled antibodies
or antibodies labeled nominally via bifunctional chelates has
added an unnecessary complication to the already complicated
experiment involving the search for the ideal diagnostic or
therapeutic antibody.
Since the First Conference many new labeling techniques and
chelating agents have been developed. One purpose of this
conference is to investigate these new techniques and chelating
agents to determine if radiochemically pure antibodies have
been developed.
References
1. Zalutsky, M. R., and Narula, A. S. A method for the radiohalogenation of
proteins resulting in decreased thyroid uptake of radioiodine. Int. J. Appi.
Radiât.Isot., 38: 1051-1055. 1987.
2. Ali, S. A., Warren, S. D., Richter. K. Y., Badger. C. C.. Eary. J. F., Press,
O. W., Krohn, K. A., Bernstein, I. D., and Nelp, W. B. Improving the tumor
retention of radioiodinated antibody: aryl carbohydrate adducts. Cancer Res.,
(Suppl.), 50: 783s-788s, 1990.
3. Kasina, S., Vanderheyden, J. L., and Fritzberg. A. R. Application of diamide
dimercaptide NjS2 bifunctional chelating agents for "Te labeling of pro
teins. J. Labeled Compos. & Radiopharm.. 23: 269-271. 1986.
4. Arano, Y., Yokoyama, A., Magata, Y.. Saji. H.. Horiuchi, K., and Torizuka.
K. Synthesis and evaluation of a new bifunctional chelating agent for ""Te
labeling proteins: p-carboxyethylphenyl-glyoxal-di(iV-methylthiosemicarba-
zone). Int. J. NucÃ-.Med. Biol.. 12: 425. 1985.
5. Tolman, G. L., Hadjian, R. A.. Morelock, M. M., Jones, P. L., Neacy. W.,
Liberature. F. A.. Sands. H.. and Gallagher. B. In vivo tumor localization of
Technetium-labeled metallothionein/monoclonal
antibody conjugates. J.
NucÃ-.Med.. 25: 20. 1984.
6. Burchiel, S. W., Hadjian. R. A.. Hladik. W. B., Gregorio, S. N., Tolman, G.
L.. Drozynski, C. A., Covell. D., Day. P. A.. Christie, J. H.. and Gallagher,
B. M. Biodistribution and pharmacokinetic analysis of "Tc-metallothionein
conjugated to B 72.3 antibodies in rhesus monkeys. J. NucÃ-.Med.. 27: 896,
1986.
7. Lanteigne, D., and Hnatowich. D. J. The labeling of DTPA-coupled proteins
with 99mTc. Int. J. Appi. Radiât.Isot., 35: 617-622, 1984.
8. Child, R. L., and Hnatowich, D. J. Optimum conditions for labeling of
DTPA-coupled antibodies with technetium-99m. J. NucÃ-.Med.. 26: 293299, 1985.
9. Liang, F. H.. Virzi, F.. and Hnatowich. D. J. Serum stability and non-specific
binding of technetium-99m labeled diaminodithiol for protein labeling. NucÃ-.
Med. Biol.. 14: 555-562. 1987.
10. Araño,Y., Yokoyama. A.. Furukawa. T.. Horiuchi. K., Yahata, T., Saji. H..
Sakahara, H., Nakashima. T., Koizumi, M., and Endo. K. Technetium-99mlabeled monoclonal antibody with preserved immunoreactivity and high in
vivo stability. J. NucÃ-.Med., 28: 1027-1033, 1987.
11. Pettit, W. A., Deland. F. H.. Bennett, S. J., and Goldenberg, D. M. Improved
protein labeling with stannous tartrate reduction of pertechnetate. J. NucÃ-.
Med., 21: 59-62. 1980.
12. Huang. J. T.. Kaiszedeh. M.. and Sakimura. 1. Detection of bacterial endo
carditis with **Tc labeled antistaphylococcal antibody. J. NucÃ-.Med., 21:
783-786. 1980.
13. Rhodes, B. A., Torvestad, D. A., Breslow, K., Burchiel, S. W., Reed, K. A.,
and Austin. R. K. 99mTc labeling and acceptance testing of radiolabeled
antibodies. In: S. W. Burchiel and B. A. Rhodes. Tumor Imaging, p. 111.
New York: Masson Publishing USA, Inc., 1982.
14. Sundrehagen, E. Formation of "Te immunoglobulin G complexes free from
radionuclides. quality controlled by radioimmunoelectrophoresis.
Eur. J.
NucÃ-.Med., 7: 549-552, 1982.
15. Steigman, J., Williams, H. P.. and Solomon, N. A. The importance of the
protein sulfhydry I group in HSA labeling with "Te. J. NucÃ-.Med., 16:573,
1975.
16. Paik. C. H., Pham. L. N. B., Hong. J. J., Sahami. M. S.. Heald. S. C.. Reba,
R. C., Steigman, J.. and Eckelman. W. C. The labeling of high affinity sites
of antibodies with '""Tc. Int. J. NucÃ-.Med. Biol., 12: 3-8, 1985.
17. Paik. C. H.. Eckelman. W. C.. and Reba. R. C. Transchelation of "Tc from
low affinity sites to high affinity sites of antibody. NucÃ-.Med. Biol., 13: 359362, 1986.
18. Fritzberg, A. R. Advances in "Tc-labeling of antibodies. NucÃ-.Med., 26:112, 1987.
19. Franz. J.. Volkert. W. A.. Barefield. E. K., and Holmes, R. A. The production
of technetium-99m-labeled conjugated antibodies using a cyclam-based bi
functional chelating agent. J. NucÃ-.Med. Biol.. 14: 569-572. 1987.
20. Fritzberg. A. R., Abrams. P. G.. Beaumier. P. L., Kasina, S., Morgan, A. C.,
Rao, T. N., Reno, J. M.. Sanderson. J. A.. Srinivasan. A., and Wilbur. D. S.
Specific and stable labeling of antibodies with technetium-99m with a diamide
dithiolate chelating agent. Proc. Nati. Acad. Sci. USA, 85: 4025-4029, 1988.
782s
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Development of Radiochemically Pure Antibodies
W. C. Eckelman
Cancer Res 1990;50:780s-782s.
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