Proc. Nati. Acad. Sci. USAVol. 85, pp. 4025-4029, June
1988Medical Sciences
Specific and stable labeling of antibodies with technetium-99m
witha diamide dithiolate chelating agent
(radioimmunoimaging/active ester/preformed chelate conjugation
to antibody fragments)
ALAN R. FRITZBERG*, PAUL G. ABRAMS, PAUL L. BEAUMIER, SUDHAKAR
KASINA, A. CHARLES MORGAN,T. N. RAO, JOHN M. RENO, JAMES A.
SANDERSON, A. SRINIVASAN, D. SCOTT WILBUR,AND JEAN-LUC
VANDERHEYDENNeoRx Corporation, 410 West Harrison Street, Seattle,
WA 98119
Communicated by Lewis H. Sarett, January 25, 1988
ABSTRACT Technetium-99m labeling of antibodies hasbeen
suboptimal because of low affinity adventitious binding,nonspecific
labeling, and loss of immunoreactivity. The dia-mide dithiolate
ligand system (N2S2) forms highly stable,well-defined tetradentate
complexes with Tc(V). Antibodiesand their fragments have been
labeled by conjugation ofpreformed
"mTc4,5-bis(thioacetamido)pentanoate active es-ter to protein amine
groups to give a chemically known99"Tc-N2S2 complex covalently
linked to antibody. Evalua-tions of the "'Tc-N2S2-bound antibodies
and their fragmentshave shown high stability and retained
immunoreactivity.
Successful targeting ofdiagnostic radionuclides to tumors
notonly provides a tool to diagnose and stage cancer but
alsodemonstrates feasibility for therapy where ligand systemscan be
applied to therapy radionuclides. Early studies withradiolabeled
antibodies utilized radioiodine (1231/1311) be-cause of extensive
experience in protein radioiodination,covalent attachment, and
ready availability of the radio-nuclide (1, 2). Improved
tumor-to-nontumor ratios wereachieved with 1"'In compared to 131I
by using diethylenetria-minepentaacetate (DTPA) bifunctional
chelating agent tech-nology (3-5). 1311 and 1"'In have long
half-lives of 8 days and67 hr, respectively. Recent studies
utilizing antibody frag-ments have shown that tumor uptake and
background tissueclearance can take place more rapidly with
fragments,allowing the use of shorter half-life radionuclides such
as99mTc (6).99mTc is the ideal diagnostic single-photon
radionuclide
with a 6-hr half-life, 140 keV 'y radiation, no
particulateradiation, and inexpensive, convenient availability.
Theseattributes allow the routine administration of doses of 30
mCi(1 Ci = 37 GBq) that result in high photon-flux
levelsfacilitating lesion detection by single-photon-emission
com-puterized tomography. Despite these attractive aspects,
thenumerous oxidation levels available and the proclivity of99mTc
for nonspecific binding to proteins preclude easyadaptation to
antibody labeling processes developed for otherradionuclides such
as 1"'in.
Antibodies have been labeled with "mTc both directly andthrough,
bifunctional chelating agents, but with suboptimalresults. Direct
labeling involves metal binding to donor atomson the protein,
resulting in multiple binding sites of low metalaffinity (7).
Bifunctional chelating agents, including DTPA (8,9) and
bis(semithiocarbazones) (10), have been studied for99mTc labeling
but have been found to suffer from problemsof (i) nonspecific
uptake because of the requirement forseveral ligands per antibody,
(ii) adventitious binding of themetal to the protein, (iii)
formation and binding of colloids to
the antibody (11), (iv) control of the oxidation level ofTc,
and(v) low yields of specifically bound "mTc (9).We have applied a
diamide dithiolate ligand system (N2S2)
to "mTc labeling of antibody fragment. This ligand systemforms
highly stable tetradentate complexes with Tc (CmTc-N2S2) at the +5
oxidation level (12). Studies of a largenumber of compounds
containing the N2S2 donor groupshowed that high yields of "mTc
complexes of the predictedstructure were obtained (13). We have
shown that otherpotential metal-binding groups, such as the
carboxylategroup present in the N2S2 ligands, do not participate in
metalbinding (14). This property enables labeling of antibody
byformation of the technetium complex, then chemical conver-sion of
the carboxylate group to an active ester, and finallyconjugation of
the preformed complex to antibody. Prelimi-nary results of studies
of this approach were included in areview of 99mTc labeling of
antibodies (15).
MATERIALS AND METHODSPreparation of
99mTc-4,5-bis(thioacetamido)pentanoyl
(N2S2)-Conjugated Anti-Melanoma 9.2.27 F(ab')2 Fragment.To a
mixture of 25 1.l of 4,5-bis(benzoylthioacetamido)pen-tanoic acid
(1.0 mg/ml solution in 90% CH3CN) and 100 ,ul of1 M NaOH was added
100 mCi of sodium [99mTc]pertech-netate in 1.0 ml of saline (0.9%
NaCl). Then 1.0 mg of sodiumdithionite (0.10 ml of a freshly
prepared 10 mg/ml solution)was added, and the mixture was heated at
750C for 15 min.The pH was brought to about 6 with 0.10 ml of 1 M
HCI and0.30 ml of 0.2 M sodium phosphate buffer (pH 6.0). Then
10.0mg of 2,3,5,6-tetrafluorophenol (0.10 ml of a 100 mg/mlsolution
in 90% CH3CN) and 12.5 mg of
1-(3-dimethylami-nopropyl)-3-ethylcarbodiimide (0.10 ml of a 125.0
mg/mlsolution in 90% CH3CN) were added, and the solution washeated
at 75°C for 30 min. The resulting tetrafluorophenylactive ester
derivative of "mTc-4,5-bis(thioacetamido)pen-tanoate was purified
by loading the reaction mixture on aconditioned C18 cartridge (J.
T. Baker), washing with 2.0 mlof20% (vol/vol) ethyl alcohol/0.01 M
sodium phosphate, pH7.0, eight times, and eluting with 100% CH3CN.
The solventwas evaporated under a stream of N2. Then 0.5 ml of
the9.2.27 F(ab')2 fragment (16) at 2.5 mg/ml and 0.50 ml of 0.2M
sodium phosphate (pH 9.0) were added for conjugation.After 15 min
at room temperature, 25 mg of lysine (0.25 ml ofa 250-mg/ml
solution at pH 9.0) was added to quenchunreacted ester. The
99mTc-N2S2-9.2.27 F(ab')2 was purifiedby passage through a G-25
Sephadex column (Pharmacia)equilibrated with phosphate-buffered
saline.
Abbreviations: N2S2, diamide dithiolate chelating system;99M
Tc-N2S2-9.2.27 F(ab')2,
99mTc-4,5-bis(thioacetamido)pentanoyl-9.2.27 F(ab')2 fragments;
DPTA, diethylenetriaminepentaacetate.*To whom reprint requests
should be addressed.
4025
The publication costs of this article were defrayed in part by
page chargepayment. This article must therefore be hereby marked
"advertisement"in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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4026 Medical Sciences: Fritzberg et al.
C
HN NH
s sI IR R
R = H. CH3CO
Tc-
S S
Lo-
sodium I I1
+ 99sTCC_ dithionite 0 NXIIN N 0+O~~m~cO~ I o 4-)
S S
2,3,5,6-tetrafluorophenol,carbodiimide
0 F F0~~~~~~~~~0~~~~~~~~~
)L NH r'vAb
Ab-NH 0 I/ F F-0
FIG. 1. Synthesis of "mTc-N2S2-antibody by formation of
9'Tc-4,5-bis(thioacetamido)pentanoate, which is converted to an
active esterby water-soluble carbodiimide and finally conjugated to
antibody via acylation of amino groups.
An idiotypic antibody (NR-2-AD) specific to an individu-al's
human B-cell lymphoma (17) was used in F(ab')2 form asa control
antibody for the in vivo biodistribution studies. Itwas labeled as
described for 9.2.27 F(ab')2.
Evaluation of ""'Tc-N2S2-9.2.27 F(ab')2 Stability. Thestability
of the 9'9Tc binding to the antibody fragment wastested by
incubation at 37TC in the presence of variouschallenge agents,
including human serum, 10 mM DTPA, 10mM
2,3-bis(thioacetamido)propanoate, and 6 M urea. Sam-ples were
removed at various times and analyzed by HPLC,isoelectric focusing
electrophoresis, and NaDodSO4/PAGE.
Radiolabeled Cell Binding Assay. The immunoreactivity
of"mTc-N2S2-9.2.27 F(ab')2 was assessed in an antigen-excesscell
binding assay (18). Radioactivity bound to cells wasdetermined at
increasing FMX-met (metastatic; ref. 19)melanoma cell levels until
maximum binding was shown(antigen excess); then correction was made
for nonspecificbinding by blocking of radiolabeled binding with
excessunlabeled antibody.In Vivo Evaluation of ""'Tc-Labeled
Antibody F(ab')2 Frag-
ments in Tumored Nude Mice. Nude mice (mean weight, 28.2g + 1.7
SD) were subcutaneously implanted with humanmelanoma xenograft
A-375 mi/m in the left flank or side.Imaging and biodistribution
studies were conducted in groupsof three and six mice,
respectively, with 9'9Tc-labeledF(ab')2 fragments of
melanoma-specific 9.2.27 antibody andnonspecific NR-2-AD antibody.
Tumor weights were 335 mg+ 185 SD for the 9.2.27 F(ab')2
biodistribution and 222 + 67mg for the NR-2-AD F(ab')2. Tumor
weights were 619 mg forthe 9.2.27 F(ab')2 and 543 mg (range,
543-720 mg) for theNR-2-AD F(ab')2 imaging studies. Imaged mice
each re-ceived 50 ,ug of protein labeled with 1-1.5 mCi in
anintravenous administration of 100 ,u via the tail vein. Micewere
imaged serially for 16 hr after injection with a
small-field-of-view (200 mm) scintillation camera (General
Elec-tric). In the biodistribution studies, mice each received 10
,ugof protein labeled with 100 uCi in an intravenously
admin-istered dose of 100 ,ul. The animals were sacrificed 20 hr
afterinjection; blood, tumor, tail (injection site), skin, heart,
bone,lung, liver, spleen, stomach, neck (thyroid), kidneys,
andintestine were isolated, blotted when appropriate, weighed,and
assayed in a crystal scintillation detector well counter.Mean
percent injected dose per g of body weight was
calculated by comparison with radioactivity of standardsamples
of injectate.
1%
A
0
0 N\ II/N 0 F F
s s-~
FIG. 2. HPLC chromatograms ofcarboxylate (A) and active ester(B)
forms of 9mTc-4,5-bis(thioacetamido)pentanoate (A) and
its2,3,5,6-tetrafluorophenyl derivative (B). The column used was
re-versed-phase Ultrasphere ODS (5 ,Am; Beckman). Conditions:
10%6(vol/vol) CH3CN/0.01 M phosphate, pH 7.0 (A); and 34%CH3CN/0.01
M phosphate, pH 7.0 (B), at 1.0 ml/min. Pairs of peaksresult from
chelate ring epimers with respect to syn or anti alkylcarboxylate
functionality relationship to the Tc oxo group (20). Thedecreased
polarity because ofesterification is evident by the increasein
organic solvent necessary to elute the complexes.
Proc. Natl. Acad. Sci. USA 85 (1988)
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Proc. Natl. Acad. Sci. USA 85 (1988) 4027
RESULTSStarting material, [9mTc]pertechnetate, was reduced
bydithionite in basic solution in the presence of the N2S2 ligandto
give 99mTc4,5-bis(thioacetamido)pentanoate, which wasconverted to
the 2,3,5,6-tetrafluorophenyl active ester byreaction with the
phenol and water-soluble carbodiimide(Fig. 1). HPLC chromatograms
of carboxylate and activeester forms of the Tc complex are shown in
Fig. 2. Thereduced polarity of the ester allowed purification by
alow-pressure reversed-phase cartridge system. Conjugationto
antibody lysine amino groups was carried out by additionof
anti-melanoma 9.2.27 F(ab')2 and buffer to adjust the pHto 9.0.
HPLC chromatograms ofcrude product and 'mTc-N2S2-9.2.27 F(ab')2
after gel filtration purification are shown inFig. 3. The yields of
the carboxylate form of the complexwere essentially quantitative.
The ester yields were about70%. Conjugation yields ranged from 50%
to 70%o. Thus,overall yields were about 35% to 50%. Total time for
theprocedure was about 3 hr.
Support for the aminoacylation reaction to give an
amideconjugate was provided by pH dependency of the reaction(Fig.
4). The yield of 'Tc-labeled antibody was low atneutral pH and
increased as the pH increased in a mannerexpected for reaction-rate
acceleration because of increasedavailability of deprotonated amino
groups. Routinely, con-jugations were run at pH 9 as a trade-off
between yield andconcern for maintaining mild conditions with
respect to therelatively unstable F(ab')2 fragments.The stability
of the 99mTc label was evaluated by challenge
under several conditions. These included human serum,chelating
agents 10 mM DTPA and 10 mM 2,3-bis(thioace-
A
B
C
FIG. 3. HPLC chromatograms of starting 9.2.27 F(ab')2
(spec-trophotometric trace in A), crude 9'Tc conjugation product
(radio-metric trace in B), and purified 9I1Tc-N2S2-9.2.27 F(ab')2
(radio-metric trace in C). The column used was Zorbax GF-250
(DuPont).Conditions: 0.2 M sodium phosphate (pH 7.0) at 1.0
ml/min.Purification was by G-25 gel filtration.
CD
86an
55F
45~
35F
25[
15
7.0 7.5 8.0 8.5pH
9.0 9.5 10.0
FIG. 4. The dependency of the reaction of the
2,3,5,6-tetrafluorophenyl active ester derivative of
FmTc-4,5-bis(thioaceta-mido)pentanoate with 9.2.27 Fab antibody
fragment on pH. Theconjugation reaction was carried out at 1.0
mg/ml antibody fragmentconcentration with pH buffered as shown by
phosphate. Nonspecificassociation of the active ester complex was
minimized by addition of25 mg of lysine after 30 min at room
temperature.
tamido)propanoate as the free thiol form of the propanoateform
of the N2S2 ligand, and 6 M urea for denaturation of theantibody
fragment. The challenge with the propanoate formof the ligand
allowed identification of released "mTc radio-activity as (i)
non-N2S2-associated, (ii) propanoate N2S2-chelated if exchangeable
to an N2S2 complex, or (iii) simplynonspecifically associated
pentanoate N2S2, the ligand formused for labeling. The results in
Table 1 show insignificantlosses of 'mTc radioactivity under all
challenge conditionsand, thus, high stability of the 99mTc label
consistent with themetal bound in a stable complex and the complex
covalentlyattached to the antibody.The effects ofantibody
derivatization by preformed 'mTc-
N2S2 (pentanoate form) complex conjugation on 9.2.27F(ab')2 were
assessed by isoelectric focusing electrophoresis(Fig. 5). The
protein mass was detected by Coomassie bluestain, and radioactivity
was detected by autoradiography. Nochange in migration after
labeling was seen in bands bystaining. However, autoradiography
indicated a shift to lowerpI of the radiolabeled species. The
covalent attachment of99mTc would be expected to give a charge
shift because of thenet -1 charge on the 99mTc-N2S2 complex.
Immunoreactivity was assessed by measuring the fractionof 'mTc
radioactivity bound to FMX-met melanoma cells.With 'mTc-N2S2
(pentanoate form) attached to whole9.2.27, a mean immunoreactivity
of 77 + 4.0% SD (6preparations) was found. Immunoreactivity
appeared to be
Table 1. Radiolabel stability studies of 99mTc-N2S2-9.2.27
F(ab')2by challenge with various agents
99MTc-N2S2 bound to antibody afterinjection, %
Challenge 1 hr 3 hr 9 hr 24 hrControl 98.0 97.8 97.4 9.7C3N2S2*
97.0 97.0 %.2 94.7DTPA 98.1 98.1 97.2 96.5Serumt 100.0 100.0
100.0Urea 98.1 98.2 97.5
*N2S2 propanoate form.tSerum stability was studied with a
separate preparation. No changewas observed in 24 hr by HPLC,
NaDodSO4/PAGE, or isoelectricfocusing techniques.
0,
Medical Sciences: Fritzberg et al.
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Proc. Natl. Acad. Sci. USA 85 (1988) 4029
defined chemistry. The chemistry involves forming a
stablecomplex with "mTc and subsequently conjugating the com-plex
by using the standard active-ester approach under mildconditions.
This has been done with a diamide dithiolateligand system. High
stability and retention of immunoreac-tivity has been demonstrated
for 9'9Tc-labeled antibodiesand their fragments by this approach.
Cleavage of F(ab')2 toFab' was not seen, which has been problematic
for otherapproaches that involved the reducing agent stannous
ion(23). Other work in our laboratory has shown similar
labelingresults for Fab or Fab'. The challenge studies of
antibodyfragments labeled with the N2S2 complexes indicate that
thelabel was stably bound. Together with rapid clearance ofantibody
fragments and lack of normal tissue accumulation,optimal imaging
can be achieved within periods appropriatefor the short half-life
of the isotope. Encouraging results ofimaging sites of metastatic
melanoma in patients by using the99'TcN2S2 preformed chelate
approach have been reported(24).Rhenium has virtually identical
structural properties in its
complexes compared to Tc (25). We have demonstrated thiswith
penicillamine, an N,S ligand (26). Application of theN2S2-metal
complex conjugation technology for radiother-apy is thus apparent
from these studies, since isotopes ofrhenium, "8Re and "mRe, have
favorable therapy propertiesbecause oftheir radiations. Preliminary
studies of '"Re and18Re indicate that the same N2S2 diamide
dithiolate tech-nology is applicable to the rhenium labeling of
proteins andgives biodistribution properties that are virtually
identical tothose of 99mTc (27).
The efforts of the NeoRx technological staff of both the
chemistryand immunology divisions in carrying out the research are
greatlyappreciated. The support of National Institutes of Health
Grant CA40528 to A.R.F. and National Cancer Institute Contract
N01-CM-67719 are gratefully acknowledged.
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