Download High-Specific-Activity 111ln-labeled

ICANCER RESEARCH
45, 5694-5699,
November 1985]
High-Specific-Activity
111ln-labeled Anticarcinoembryonic Antigen
Monoclonal Antibody: Improved Method for the Synthesis of
Diethylenetriaminepentaacetic Acid Conjugates1
Raymond J. Paxton, James G. Jakowatz, J. David Beatty, Barbara G. Beatty, William G. Vlahos,
Lawrence E. Williams, Brian R. Clark, and John E. Shively2
Division of Immunology, Beckman Research Institute of the City of Hope [R. J. P., J. E. S.], Department of General Oncologie Surgery [J. G. J., J. D. B., B. G. B.,
W. G. V.], Division of Radiological Sciences [L. E. W.], and Division of Pediatrics [B. R. C.], City of Hope National Medical Center, Duarte, California 9W10
ABSTRACT
dehalogenation are disadvantages of the method. In nude mouse
xenograft studies using 131l-labeled monoclonal antibodies to
A new method has been developed for conjugating diethylenetriaminepentaacetic acid (DTPA) to proteins using the Nhydroxysuccinimide active ester of DTPA. The DTPA-active ester
was prepared using diisopropylcarbodiimide in a
step synthesis. DTPA-conjugated proteins were
adding the DTPA-active ester reaction mixture to
tions (5 mg/ml) buffered at pH 7.0 and purified by
simple single
prepared by
protein solu
Sephadex G-
50 chromatography. A monoclonal antibody directed against
carcinoembryonic
antigen was reacted with four different
amounts of the DTPA-active ester. Solid-phase enzyme immunoassay showed that the ¡mmunologicalactivity of the antibody
conjugate was not altered when the active esterantibody molar
ratio was 36:1 or 72:1; however, it decreased when the ratio
was 180:1 or 360:1. The antibody heavy and light chains had
slightly decreased electrophoretic mobilities when analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a
result consistent with the covalent attachment of DTPA to the
protein. Sephadex G-200 chromatography showed that the na
tive and conjugated antibodies were the same size. When the
DTPA-conjugated antibody was incubated with 10, 50, and 100
/¿Ci
of "Mn/^g of protein, specific activities of 9.8, 43.1, and 56.3
nC'\/u.g were obtained. Enzyme immunoassay and radioimmunoassay of the '"In-labeled antibody showed that it retained its
full immunological activity. The high specific activity of the 111ln-
CEA Haskell ef al. (3) and Hedin et al. (4) demonstrated tumor
localization with little liver and spleen uptake. The penetration of
the antibody into tumors and therefore the tumor to blood ratios
were improved by the use of F(ab) or F(ab')2 antibody fragments
(5, 6). The use of 131l-labeledanti-CEA polyclonal antibodies has
required sophisticated background subtraction techniques in
order to reliably detect tumors in humans (7, 8). In a human
study by Mach ef al. (9) the use of monoclonal antibodies (to
another colorectal antigen) or their fragments gave equivalent
results and also required background subtraction.
The use of111In as a label has the advantages that it can be
linked irreversibly to proteins via a bifunctional chelating agent
such as derivatives of DTPA, has more favorable emission
probability than 131I,does not emit /3-radiation, and has a short
half-life (67 h). Progress in the use of '"In-labeling has depended
upon the development of appropriate bifunctional chelating
agents. A commonly used reagent is the symmetrical bicyclic
anhydride of DTPA prepared according to Hnatowich ef a/. (1012) and Layne ef al. (13). Anti-CEA labeled with 1"ln by this
method gave substantial spleen and liver uptake in addition to
the expected tumor uptake in the nude mouse system (10). A
similar result was reported by Halpern ef al. (14) utilizing a mixed
anhydride reagent for conjugating DTPA to anti-CEA. The ac
cumulation of the radiolabeled antibody in the liver and spleen
has not been explained but it is potentially due to the modification
of the antibody by the conjugating reagents. Paik ef al. (15, 16)
showed that both the symmetrical bicyclic anhydride of DTPA
and the mixed anhydride of DTPA led to decreased antibody
activity when large reagent to protein ratios were used. When
increasing amounts of the symmetrical bicyclic anhydride were
reacted with antibody, increasing amounts of cross-linked protein
labeled antibody makes it suitable for imaging carcinoembryonic
antigen-bearing tumors using low doses of antibody.
INTRODUCTION
In vivo detection of tumors can be accomplished with a variety
of radiolabeled antibodies. Early studies utilized antibodies
species were observed (15). At the suggested lower ratios of
against CEA3 radiolabeled with 131I(1, 2). Although antibodies
anhydride to protein the specific activities achieved with '"Incan be labeled to high specific activities (>10 mCi/mg) by radioiodination with 131I,the long half-life (8 days), undesirable ß- labeling were low (1-4 ¿¿Ci/^g).
Initial work in our laboratory confirmed the problems associ
radiation, and loss of the label from the antibody by tissue
ated with the mixed anhydride and symmetrical bicyclic anhy
dride reagents. An alternate method of conjugating DTPA to
1This work was supported by Grant CA37808 from the National Large Bowel
proteins involves activation of only one carboxylic acid group per
Cancer Project (J. E. S.) and an Arthur R. Mehr Research Fellowship (J. D. B.).
DTPA molecule using the A/-hydroxysuccinimide active ester.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: CEA, carcinoembryonic antigen; MAB, monoclonal
antibody; DTPA, diethylenetriaminepentaacetic
acid; BSA, bovine serum albumin;
DTPA-OSU. N-hydroxysuccinimide
ester of diethylenetriaminepentaacetic
acid;
anti-CEA MAB-DTPA. DTPA-conjugated anti-CEA monoclonal antibody; BSADTPA, DTPA-conjugated BSA; ELISA, enzyme-linked immunosorbent assay; antiCEA MAB-DTPA-1"ln, DTPA-conjugated anti-CEA monoclonal antibody labeled
with '"In; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
While this study was in progress, a preliminary account of a
similar method was described by Buckley and Searle (17). In this
report, we present an improved protocol for conjugating DTPA
to proteins. Antibody conjugates prepared using this method
had higher specific activities when labeled with 1"ln than was
previously reported and retained full immunological activity. The
Received 3/15/85; accepted 7/12/85.
CANCER RESEARCH
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1985
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111ln-LABELED ANTI-CEA
use of the 111In-labeled antibody in a nude mouse system is
MAS SYNTHESIS
Apple II Plus computer. Quantitäten of anti-CEA activity was performed
according to Beatty ef al. (22). ELISA of anti-CEA MAB-DTPA and antiCEA MAB-DTPA-1 "In were performed in the same fashion.
described in the companion paper (18).
Electrophoresis. DTPA-conjugated proteins were analyzed by SDSpolyacrylamide gel electrophoresis, as described by Laemmli (23). Sam
ples were boiled in dissociation buffer (0.0625 M Tris-HCI, pH 7.0, 6%
SDS, 20% glycerol, and 5% 2-mercaptoethanol) for 5 min and applied to
MATERIALS AND METHODS
Reagents. The following reagents were used: DTPA, diisopropylcarbodiimide, and triethylamine (Aldrich Chemical Co., Milwaukee, Wl); Nhydroxysuccinimide,
BSA, Sephadex G-50 and G-200, and protein ASepharose CL-4B (Sigma Chemical Co., St. Louis, MO); sodium acetate
10% acrylamide slab gels. Proteins were visualized with Coomassie blue
stain. DTPA-conjugated proteins labeled with 1"ln were analyzed using
the same method except that 1 mw dithiothreitol replaced the 2-mercap
toethanol in the dissociation buffer and the samples were boiled for 3
min. '"In-labeled proteins were visualized by autoradiography with Ko
(Puratronic grade) and acetic acid (ultrapure) (Alfa Products, Danvers,
MA). Acetonitrile (high performance liquid chromatography grade; J. T.
Baker Co., Phillipsburg, NJ) was dried over molecular sieves and trieth
ylamine was distilled from ninhydrin and calcium hydride before they
were used.
The previously described anti-CEA monoclonal antibody (19,20), clone
T84.66 A3.1 H11, was purified from mouse asertes fluid by protein ASepharose chromatography (21). Anti-cytochrome
P-450 monoclonal
antibody was obtained from Wayne Levin, Hoffmann-La Roche Inc.,
Nutley, NJ. 111lnCI3(research grade, 5 mCi/0.1 ml in dilute HCI) was a
gift from Medi-Physics, Inc. (Emeryville, CA).
Synthesis of DTPA Af-Hydroxysuccinimide
dak X-OMAT AR film (Eastman Kodak Co., Rochester, NY).
Mass Spectrometry. The mass spectra of the DTPA-OSU reaction
mixture were obtained using a JEOL JMS-HX100 mass spectrometer
equipped with a fast atom bombardment ion source. Samples were
dissolved in glycerol and applied to a 2- x 5-mm stainless steel sample
stage. Spectra were acquired on a JEOL DA5000 data system.
RESULTS
Ester. DTPA (12 mg,
Preparation of BSA-DTPA and Anti-CEA MAB-DTPA Con
jugates. The /V-hydroxysuccinimide active ester of DTPA was
prepared by a reaction commonly used in peptide synthesis. The
DTPA-OSU active ester was not isolated; however, the reaction
mixture was examined by mass spectrometry. Fast atom bom
bardment mass spectrometry (negative ion mode) of the reaction
mixture showed the expected mass:charge ratio of 489.
The reaction of DTPA-OSU with proteins is expected to pro
duce an amide bond between the e-amino group of lysine resi
dues and the activated carboxyl group of DTPA. The modification
of proteins with this large highly charged molecule might alter
the electrophoretic mobility of the proteins in SDS-PAGE gels.
To investigate this possibility, the test protein BSA was reacted
with 1, 2, 5, and 10 /¿Iof DTPA-OSU, giving reagent.-protein
0.0305 mmol) and triethylamine (0.017 ml, 0.122 mmol) were dissolved
in 0.20 ml acetonitrile by stirring at 50°C for 1.5 h. After cooling the
solution to 25°C, A/-hydroxysuccinimide (1.76 mg, 0.0153 mmol) and
diisopropylcarbodiimide
(0.0024 ml, 0.0153 mmol) were added and the
reaction was stirred for 1.5 h. The theoretical concentration of active
ester is 0.06 M. The reaction mixture was used directly to prepare DTPAconjugated proteins.
Synthesis of DTPA-conjugated
Proteins.
Lyophilized anti-CEA
monoclonal antibody was dissolved in 0.10 M NaHCO3:0.20 ITIM EDTA,
pH 7.0 at a concentration of 5 mg/ml. DTPA-conjugated anti-CEA MAB
was prepared by adding 1, 2, 5, or 10 n\ of the DTPA-OSU reaction
mixture to 50 /j\ of the protein solution and mixing every 20 min for 1 h.
The anti-CEA MAB-DTPA conjugate was separated from free DTPA and
other reaction by-products using either a 0.7- x 30-cm Sephadex G-50fine column or a 0.7- x 50-cm Sephadex G-200 column equilibrated with
0.10 M sodium acetate, pH 5.6. BSA-DTPA and anti-cytochrome P-450
MAB-DTPA conjugates were prepared using the same methods.
Charging of DTPA-conjugated Proteins with '"In. An equal volume
of 1.0 M sodium acetate, pH 6.0 was added to "1lnCI3 (5 mCi/0.1 ml).
molar ratios (theoretical) of 16, 32, 80, and 160, respectively,
and the reaction mixtures were analyzed by SDS-PAGE. The
electrophoretic mobility of BSA decreased as the amount of
DTPA-OSU was increased (Fig. ~\A). A minor protein band with
Aliquots of this mixture (10,100, or 500 j/Ci) were incubated with 10 ^g
(0.2 mg/ml) of the anti-CEA MAB-DTPA for 2-16 h. EDTA (final concen
tration, 1 mw) was then added to complex any free 111ln.Thirty min after
adding EDTA, the efficiency of charging was examined by chromatog
raphy on a 0.7- x 30-cm Sephadex G-50-fine column equilibrated with
0.10 M sodium acetate, pH 5.O. BSA-DTPA and anti-cytochrome P-450
MAB-DTPA conjugates were charged with 111lnusing the same method.
Stability Testing. Anti-CEA MAB-DTPA (200 ng) was charged with
1"ln (10 MCÌ/M9
of antibody) as described previously. EDTA was added
to complex any free 111ln;then the '"In-labeled antibody (100 /¿g)was
added to 9.5 ml of fresh human plasma and incubated at 37°C. At 0.5,
an apparent molecular weight of 97,000 was detected when
DTPA-OSu (microliters)
O
CANCER
RESEARCH
2
5 io
O
B
DTPA-OSu (microliters)
0
1
2
5
10 0
•?2002 1,6*
97ht
68-
5,24,48, and 72 h, 1-ml aliquots were removed and analyzed by protein
A-Sepharose chromatography (21). The nonbound protein was washed
from the column with 0.1 M sodium phosphate, pH 8, and IgG was eluted
from the column with 0.1 M sodium citrate, pH 6 and 3.5. The column
fractions from the different times were saved and counted after sufficient
radioactive decay.
ELISA. Enzyme immunoassay microtiter plates (Costar, Cambridge,
MA) were coated with CEA at 5 Mg/ml, washed, and incubated with 1%
BSA to block any remaining binding sites. The plates were incubated
with serial dilutions of anti-CEA MAB, washed five times, incubated with
goat anti-mouse IgG-alkaline phosphatase conjugate (Cappel Laborato
ries, Malvern, PA), washed five times, incubated with p-nitrophenylphosphate (Sigma), and the absorbance of each well was measured at 405
nm on a Dynatek MR 580 enzyme immunoassay reader linked to an
i
43-
30-
i
Fig. 1. SDS-polyacrylamide gel electrophoresis of BSA-DTPA and anti-CEA
MAB-DTPA reaction mixtures. BSA (A) and anti-CEA MAB (B), 250 iig of each at
5 mg/ml, were reacted with the indicated amounts of DTPA-OSU(OSu)and 3 ng
of each BSA-DTPA conjugate and 5 ^g of each anti-CEA MAB-DTPA conjugate
were analyzed by SDS-PAGE.The reagentrprotein molar ratios (theoretical) were
0, 16, 32, 80, and 160 for BSA and 0, 36, 72, 180, and 360 for anti-CEA MAB,
respectively.
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111ln-LABELED ANTI-CEA
MAB SYNTHESIS
BSA was reacted with 10 pi of DTPA-OSU. The nature of this
band is not known; however, it possibly represents a uniquely
modified form of BSA which migrates anomalously in SDS-PAGE
gels (see "Discussion").
Anti-CEA MAB was also reacted with 1, 2, 5, and 10 M' of
DTPA-OSU (theoretical reagent: protein molar ratios of 36, 72,
180, and 360, respectively), and the reaction mixtures were
analyzed by SDS-PAGE. The electrophoretic mobility of both the
antibody heavy (M, 50,000) and light (M, 30,000) chains de
creased as the amount of DTPA-OSU was increased (Fig. 1ß).
The higher molecular weight forms of the light chain migrated as
discrete species easily distinguishable from the native light chain.
Minor protein bands with approximate molecular weights of
80,000,120,000, and 200,000 were detected when the antibody
was reacted with 10 ^l DTPA-OSU and may represent crosslinked heavy and light chains (see "Discussion").
Purification and Characterization of Anti-CEA MAB-DTPA
Conjugates. Anti-CEA MAB-DTPA conjugates were separated
from free DTPA and other reaction by-products by Sephadex G50 chromatography (Chart 1). Slow flow rates (<2 ml/h) improved
resolution of the two peaks and prevented free DTPA from
contaminating the antibody conjugates. The use of sodium ace
tate buffer allowed the anti-CEA conjugates to be charged with
111In immediately after chromatography (see below).
Anti-CEA conjugates were also purified by Sephadex G-200
chromatography. Chart 2 shows that the anti-CEA conjugate
eluted from the column in exactly the same volume as did the
unmodified anti-CEA MAB. This result suggested that the size
of the native and conjugated antibodies were the same, a result
consistent with the SDS-PAGE analysis.
Purified anti-CEA MAB-DTPA conjugates were tested for immunological activity using a solid-phase enzyme immunoassay.
The ELISA results (Table 1) show that the anti-CEA conjugates
prepared with 1 or 2 n\ of DTPA-OSU retained 100% of their
.3
.2
4
8
12
162024283236
FRACTION
Chart 2. Sephadex G-200 chromatography of anti-CEA MAB and anti-CEA
MAB-DTPA conjugate. Anti-CEA MAB (750 ^g) and an anti-CEA MAB-DTPA
conjugate reaction mixture (750 ^g; theoretical reagent:protein molar ratio of 72)
were chromatographed separately on a 0.7- x 50-cm Sephadex G-200 column
equilibrated with 0.1 M sodium acetate, pH 5.6. Fractions of 0.5 ml were collected
and their absorbance at 280 nm was determined. Fractions 30-35 of the anti-CEA
MAB-DTPA cnromatogram contained the by-products of the conjugation reaction.
•.anti-CEA MAB; O, anti-CEA MAB-DTPA.
Table 1
Immunological activity of anti-CEA MAB-DTPA conjugates
Activity was measured in an ELISA in which CEA-coated plates were incubated
with anti-CEA MAB or its DTPA conjugate. Bound MAB was detected with alkaline
phosphatase-conjugated second antibody. The antibody conjugates were prepared
by adding the indicated amounts of DTPA-OSU (0.06 M) to a constant amount of
antibody (501¡\,5 mg/ml) and were purified by Sephadex G-50 chromatography.
DTPA-OSU
% anti-CEA antibody
activity
0
1(36)"
100
100
2(72)
100
37
5(180)
10(360)
9
* Numbers in parentheses, reagent:protein molar ratios (theoretical).
.5
.4
immunologlcal activity, whereas the conjugates prepared with 5
and 10 ¿¿I
of DTPA-OSU showed a decrease in immunological
activity. All subsequent experiments were done with anti-CEA
MAB-DTPA conjugates prepared with 2 /¿I
of DTPA-OSU (theo
8
I2
I6
20
24
28
FRACTION
Chart 1. Sephadex G-50 chromatography of an anti-CEA MAB-DTPA conjugate
reaction mixture. Anti-CEA MAB-DTPA (250 ^g) was prepared by reacting the
antibody (5 mg/ml) with 2 ¡Aof DTPA-OSU (theoretical reagent:protein molar ratio
of 72). The reaction mixture was applied to a 0.7- x 30-cm Sephadex G-50 column
equilibrated with 0.1 M sodium acetate, pH 5.6. Fractions of 0.5 ml were collected
and their absorbance at 280 nm was determined. Fractions 10-13 contained the
antibody conjugate and Fractions 22-27 contained the reaction by-products.
CANCER
RESEARCH
retical reagentprotein molar ratio of 72:1).
Charging of BSA-DTPA and Anti-CEA MAB-DTPA Conju
gates with 111ln.BSA-DTPA conjugates (10 ng) were incubated
with either 500 or 1000 /iCi of 111ln.EDTA was added to complex
any free 111ln and the efficiency of charging was tested by
Sephadex G-50 chromatography. Table 2 shows that the specific
activity of BSA increased as the amount of DTPA-OSU increased.
Anti-CEA MAB-DTPA conjugates prepared with 2 n\ of DTPAOSU were incubated with 111ln. EDTA was added to complex
any free 111ln and the efficiency of charging was tested by
Sephadex G-50 chromatography. In the example shown in Chart
3, 89% of the 111lneluted with the protein peak. When unconjugated anti-CEA MAB was charged with 111ln, 0% of the 111ln
eluted with the protein peak (data not shown). Table 3 contains
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'"In-LABELED
ANTI-CEA
Table2
by solid-phase enzyme immunoassay
Charging of BSA-DTPA conjugates with 111/n
ratio
OiCi/Mg)50
and radioimmunoassay
(Chart 4). When the results of the assay systems were compared
at two levels of charging with 111ln equivalent results were
BSA-DTPAconjugates were prepared by adding the indicatedamountsof DTPAOSU (0.06 M)to a constant amount of BSA (50 ¡A,
5 mg/ml). After purification by
gel chromatography, BSA-DTPAconjugates (10 /ig) were incubated overnight with
either 500 or 1000 ¿iCi
of "'In. EDTA was then added to complex free '"In and
the "'In-labeled conjugates were purified by gel chromatography.
DTPA-OSUd¿)
used to prepare
conjugate1
(16)a
MAB SYNTHESIS
obtained. The curves were parallel when comparing unconjugated versus DTPA-conjugated antibody in the EIA. This result
suggested that the DTPA-conjugation had little effect on antibody
activity.
Several attempts were made to analyze111In-labeled anti-CEA
conjugates by SDS-PAGE. If standard conditions were used, no
radioactivity was associated with the antibody heavy and light
chains. When 1 mw dithiothreitol was used in place of 5% (0.71
M) 2-mercaptoethanol in the sample dissociation buffer, both the
activity
(i-Ci/Mg)21.6
2(32)
38.4
50
48.4
5(80)
50
10(160)
47.3
50
5(80)
100
85.1
10(160)Charging
100Specific
92.7
" Numbers in parentheses, reagent:protein molar ratios (theoretical).
heavy (M, 50,000) and light chains (M, 30,000) were radioactively
labeled (Fig. 2).
Stability of "'In-labeled Anti-CEA MAB Conjugates. To
measure the stability of the anti-CEA MAB-DTPA-111In complex,
the labeled antibody was incubated with fresh human plasma
and analyzed by protein A-Sepharose chromatography. The
amount of 111ln-labeledantibody bound to the column decreased
.10
.08
from 95% after 0.5 h of incubation to 75% after 72 h of incubation
(Table 4). The majority of111In-labeled antibody eluted at pH 6
96
.06
72
o .04
<v
01
48 i
<o
'o
.02
"Õ
U
o—o-o o o o o g
8
12
16
20
FRACTION
24
28
Chart 3. Chargingof anti-CEA MAB-DTPAwith '"In. Anti-CEA MAB-DTPA(114
ng) and anti-CEA MAB-DTPA (114 pg) charged with '"In at 10 ¿iCi/jigwere
chromatographed separately on a 0.7- x 30-cm SephadexG-50 columnequilibrated
with 0.1 M sodium acetate, pH 5.0. Fractions of 0.5 ml were collected in both
cases. The absorbance of each fraction at 280 nm was determined for the
uncharged conjugate (•)
and the radioactivity of each fraction was determined for
the '"In-labeled conjugate (O) by counting 10-/il aliquots in a Beckman Gamma
300 radiation counter.
Tables
Charging of anti-CEAMAB-DTPAconjugates with 11t/n
Anti-CEA MAB-DTPA conjugates were prepared by adding 2 n\ of DTPA-OSU
to 50 »il
of antibody (5 mg/ml) giving a reagentrproteinmolar ratio of 72 (theoretical).
After purification by gel chromatography, antibody conjugates (10 ng) were incu
bated overnight with either 100, 500, or 1000 /iCi of '"In, treated with EDTA, and
purified by gel chromatography.
Charging
ratio10
activity9.8
efficiency98
43.1
50100Specific
86.2
56.3"
56.3.Charging efficiency equals specific activity -Charging
charging ratio.
65536 16384 4096 1024 256
inverse Dilution
the results obtained when anti-CEA MAB-DTPA was charged
with increasing amounts of 111ln.The efficiency of charging was
98 and 86% at 10 and 50 u.C\/u.g of protein, respectively. The
charging efficiency at 100 u.C\/u.g of protein dropped to 56%
implying that the anti-CEA conjugate was approaching saturation
with 111ln.
111ln-labeled anti-CEA MAB-DTPA conjugates were analyzed
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64
16
Chart 4. Simultaneous enzyme immunoassay (ELISA) and radioimmunoassay
of anti-CEA MAB and anti-CEA MAB-DTPAconjugates. Anti-CEA MAB-DTPAwas
prepared and purified as described in Chart 1. ELISA was performed as described
in Table 1. After the absorbance of each well was determinedat 405 nm, the wells
were separated and counted. Antibody activity is expressed as the percentage of
the average of the maximum A«»
nm or maximum cpm at the plateau region. A,
ELISA (•)
and radioimmunoassay(O) of 20 pg anti-CEA MAB-DTPA + 2 pg antiCEA MAB-DTPA-'"ln (50 pCi/pg); ELISA (•)
and radioimmunoassay(D) of 20 pg
anti-CEA MAB-DTPA + 2 /ig anti-CEA MAB-DTPA-'"ln (10 nCijug); B, ELISA (•)
and radioimmunoassay(O) of 20 pg anti-CEA MAB + 2 pg anti-CEA MAB-DTPA'"In (50 pC\/pg); ELISA (•)
and radioimmunoassay(O) of 20 ^g anti-CEA MAB +
2 pg anti-CEA MAB-DTPA-'"ln (10 pC\/pg).
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'"In-LABELED
ANTI-CEA
MAB SYNTHESIS
Stability of '"In-labeled
Table 4
anti-CEA MAB-DTPA conjugate
Anti-CEA MAB-DTPA was charged with '"In, treated with EDTA to complex
any free '"In, incubated with fresh human plasma for different times, and analyzed
by protein A-Sepharose chromatography as described in "Materials and Methods."
The percentage of '"In-labeled antibody bound to the column was then calculated.
10-3)8Incubation
Column fraction
(cpm x
antibody
(%)BA
bound to column
CA +
time
(h)0.5
B95
+
+ +96
B
144
1,607
320
92
93
5
1,639
250
24
309
84
86
48
419499B1,710
1,577
231
7975B
8178C
72A85
1,458C345 266'"In-labeled
" Fraction A, radioactivity not bound to column plus 0.1 M sodium phosphate
wash, pH 8. The radioactivity due to tree '"In (EDTA-complex) has been subtracted;
Fraction B, radioactivity eluted with 0.1 M sodium citrate, pH 6; Fraction C,
radioactivity eluted with 0.1 M sodium citrate, pH 3.5.
H
mixed anhydride reagent has been used successfully; however,
several parameters must be carefully controlled to conjugate
efficiently the antibody with DTPA while retaining antigen-binding
*
activity (16).
Problems with both of these methods prompted us to develop
a new method for conjugating DTPA to proteins. The stoichiometry of reagents used in preparing the DTPA-OSU active ester
(DTPA:A/-hydroxysuccinimide:diisopropylcarbodiimide,2:1:1)
was chosen so as to minimize the potential for producing a diactivated species of DTPA. The mass spectral results indicated
that only a mono-activated species of DTPA was present in the
reaction mixture. While this study was in progress, the synthesis
of the same reagent was reported by other investigators using
different reaction conditions (17).
Initially BSA was used to test the effectiveness of the reagent.
The change in mobility of BSA on SDS-PAGE gels clearly showed
that increasing amounts of DTPA-OSU caused an increased
modification of the protein (Fig. ~\A).The decreased electrophoretic mobility of the modified protein was in agreement with other
studies (24) which showed that treatment of proteins with succinic and maleic anhydrides caused a similar decrease in electro-
Fig. 2. SDS-polyacrylamide gel electrophoresis of '"In-labeled antibody. AntiCEA MAB-DTPA (1 ng) was charged overnight with '"In (1 /¿Ci)
and analyzed by
SDS-PAGE as described in "Materials and Methods." H, heavy chain; L. light chain.
(Fraction B), consistent with its being an lgG1. When the pH 3.5
eluent (Fraction C) was included in the calculations, the amount
of labeled antibody retained on the column was only slightly
higher. The experiment described by Table 4 was repeated,
except that the free 111ln(EDTA-complex) was first removed by
gel filtration chromatography. The decrease in 111In-labeled anti
phoretic mobility. At the highest reagent:protein ratio tested, a
trace amount of a new protein species (M, ~ 97,000) was
observed. This molecular weight is clearly less than that ex
pected for a BSA dimer (Mr ~ 136,000) and most probably
represents a specific modification of BSA distinct from the ma
jority of the protein. One possibility is that an intramolecular
cross-link has occurred due to the formation of a di-activated
species of DTPA-OSU. We have reduced this probability by
utilizing an A/-hydroxysuccinimide:DTPA ratio of 0.5:1.0.
After being purified by Sephadex G-50 chromatography, the
BSA-DTPA conjugates were charged with 111ln.The results of
Table 2 show that the increase in specific activity of the 111ln-
DISCUSSION
labeled BSA-DTPA conjugates paralleled the increase in the
amount of DTPA-OSU used to prepare the conjugates. Therefore
it can be concluded that the reagent does produce a covalent
attachment of DTPA to the protein. The increase in specific
activity also paralleled the decrease in electrophoretic mobility of
the BSA-DTPA conjugates (Fig. ~\A).This further suggests that
Several methods are currently available for the preparation of
DTPA-conjugated antibodies. The symmetrical bicyclic anhydride
of DTPA is easy to use, but because it is a bifunctional reagent,
the potential for antibody cross-linking is present (15). The DTPA-
the decreased electrophoretic mobility was due to the covalent
attachment of DTPA to the protein.
Anti-CEA MAB-DTPA conjugates were prepared using the
same method. The SDS-PAGE results for the antibody conju-
body bound to the column was similar, from 93% at 0.5 h to
80% at 72 h.
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'In-LABELED
ANTI-CEA
gates (Fig. ÃŒB)
were similar to those for the BSA conjugate (Fig.
1A). The antibody heavy (M, 50,000) and light (M, 30,000) chains
showed a decrease in electrophoretic mobility as the amount of
DTPA-OSU increased. Trace amounts of higher molecular weight
species were present at the highest reagentprotein molar ratio.
Species consistent with the observed molecular weights are HL,
H2, and H2L2 complexes (H, heavy chain; L, light chain). Since
the two heavy and two light chains are in close contact in the
IgG molecule, cross-linking between heavy and light chains could
have occurred if a di-activated species of DTPA-OSU were
present.
After purification, the antibody conjugates were tested for
immunological (antigen-binding) activity. Table 1 shows that 2 ¡i\
of DTPA-OSU could be used without decreasing the immunolog
ical activity of the antibody. Theoretically this amount gives a
DTPA-OSU:antibody molar ratio of 72:1. The actual ratio may be
less than this due to incomplete formation and/or hydrolysis of
the reagent. In comparing the results of Fig. 1B with Table 1, it
can be seen that the loss of immunological activity with 5 and
10 M' of DTPA-OSU was accompanied by an increase in the
amount of protein modification. With 2 /tl of DTPA-OSU, there
was only a slight amount of protein modification and the higher
molecular weight species were not observed.
Anti-CEA MAB-DTPA conjugates were charged with 111lnand
analyzed by gel filtration chromatography. A specific activity of
43.1 nC\/tig was obtained at a charging ratio of 50 fiC\/ng. This
specific activity was substantially higher than those reported
previously (10,11,14,17).
We have also tested the immunolog
ical activity of 111ln-labeled antibody conjugates. Chart 4 shows
that '"In-labeled conjugates had antigen-binding properties sim
ilar to either conjugated or unconjugated antibody. Therefore the
presence of 1l1ln had no measured effect on the antibody activity.
The stability of the anti-CEA MAB-DTPA-1 "In complex was
tested by incubating it with fresh human plasma. The amount of
labeled antibody bound to protein A-Sepharose decreased from
95 to 75% after a 72-h incubation. Using a DTPA-conjugated
antibody prepared with the mixed anhydride of DTPA, Halpern
ef al. (14) reported an 89 to 73% decrease in the immunoreactivity of the antibody after incubating in human serum for 72 h.
Although the methods used to test the stability of the antibodyDTPA-111ln complex were different, the results of the two exper
iments were similar. These results provide further evidence that
the DTPA-OSU active ester produces a stable covalent attach
ment of DTPA of proteins.
In conclusion, we have developed and systematically tested a
new method for synthesis of DTPA-conjugated proteins. The
method is both effective and reproducible in producing high
specific activity antibodies that retain 100% of their immunolog
ical activity. The higher specific activities obtainable with this
method should be clinically useful. By using a smaller dose of
antibody, the potential for an immunogenic response is lessened.
MAB SYNTHESIS
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High-Specific-Activity 111In-labeled Anticarcinoembryonic
Antigen Monoclonal Antibody: Improved Method for the
Synthesis of Diethylenetriaminepentaacetic Acid Conjugates
Raymond J. Paxton, James G. Jakowatz, J. David Beatty, et al.
Cancer Res 1985;45:5694-5699.
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