An Improved Method for Conjugating Monoclonal Antibodies with N

This improved conjugation procedure facilitates large-scale production and radiometal labeling of cT84.66−DOTA for clinical radioimmunotherapy trial...
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Bioconjugate Chem. 2001, 12, 320−324

An Improved Method for Conjugating Monoclonal Antibodies with N-Hydroxysulfosuccinimidyl DOTA Michael R. Lewis,† Jim Y. Kao,‡ Anne-Line J. Anderson,‡ John E. Shively,*,§ and Andrew Raubitschek‡ City of Hope Graduate Program in Biological Sciences, Department of Radioimmunotherapy, City of Hope National Medical Center, and Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010. Received July 26, 2000; Revised Manuscript Received December 5, 2000

A simple, water-soluble procedure for conjugation of monoclonal antibodies to 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) has been improved by optimizing pH, buffer, and temperature conditions for the preparation of N-hydroxysulfosuccinimidyl DOTA and its conjugation to the human/murine chimeric anti-carcinoembryonic antigen antibody cT84.66. This improved method results in a 6-fold increase in conjugation efficiency, a 3-7-fold decrease in antibody cross-linking, a more homogeneous population of conjugate species, and a 5-fold decrease in the quantities of reagents needed for conjugation. The cT84.66-DOTA conjugate was labeled to high specific activity with 111In, 90 Y, 88Y, 64Cu, and 67Cu, affording near-quantitative incorporation of the majority of these radiometals. This improved conjugation procedure facilitates large-scale production and radiometal labeling of cT84.66-DOTA for clinical radioimmunotherapy trials.

INTRODUCTION

Cancerous tumors can be targeted specifically by radiolabeled monoclonal antibodies (mAbs)1 used in clinical imaging and therapy (1, 2), and a wide variety of medically useful radiometals are available for these applications. Considerable effort has been devoted to the development of bifunctional chelating agents (BCAs) for covalent attachment of radiometal ion complexes to mAbs (3-5). An important property of the BCA is that it chelates radiometals with high in vivo stability, resulting in minimal deposition of free radiometal in normal tissues (6). The macrocyclic chelating agent 1,4,7,10tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) is an attractive candidate for conjugation of radiometals to antibodies. DOTA binds a large number of metal ions with extremely high thermodynamic (7, 8) and kinetic (9, 10) stability, and mAb-DOTA conjugates have shown minimal loss of chelated radiometal in vivo (11-14). Recently, we developed a simple, water-soluble chemical method for conjugation of DOTA to proteins using commercially available reagents (15). This method involved the preferential activation of one carboxyl group of DOTA with N-hydroxysulfosuccinimide (sulfo-NHS) * To whom correspondence should be addressed. Phone: (626) 301-8301. Fax: (626) 301-8186. E-mail: [email protected]. † City of Hope Graduate Program in Biological Sciences. ‡ Department of Radioimmunotherapy. § Division of Immunology. 1Abbreviations: mAb, monoclonal antibody; BCA, bifunctional chelating agent; DOTA, 1,4,7,10-tetraazacyclododecaneN,N′,N′′,N′′′-tetraacetic acid; DOTA-OSSu, N-hydroxysulfosuccinimidyl DOTA; sulfo-NHS, N-hydroxysulfosuccinimide; EDC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; EDTA, ethylenediaminetetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; HSA, human serum albumin; IEF, isoelectric focusing; CEA, carcinoembryonic antigen; cT84.66, human/ murine chimeric anti-CEA mAb; cT84.66-DOTA, cT84.66 conjugated to DOTA using DOTA-OSSu; HACA, human antichimeric antibody.

and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and reacting the resulting DOTA active ester with proteins at pH 8.5-9.0. The systematic development of elevated temperature, optimum pH, and appropriate buffer conditions allowed rapid, efficient, and kinetically stable labeling of immunoconjugates with 111In(III) and 90Y(III) at high specific activities. In this report, we describe improvements to this method, which make practical large-scale antibody conjugation for clinical radioimmunotherapy trials and result in near-quantitative labeling yields with radioisotopes of In, Y, and Cu. EXPERIMENTAL PROCEDURES

Reagents. The antibody cT84.66, a human/murine chimeric anti-carcinoembryonic antigen (anti-CEA) mAb, isotype IgG1, κ, was prepared as described previously (16). DOTA internal salt was purchased from Parish Chemical Co. (Orem, UT). Sulfo-NHS and EDC were obtained from Pierce (Rockford, IL). Cobalt powder (99.995%) was obtained from Aldrich (Milwaukee, WI). Chelex 100 (Biotechnology Grade, 100-200 mesh, sodium form) was purchased from Bio-Rad (Hercules, CA), and buffers used for radiolabeling reactions were passed over a Chelex 100 column (1 × 15 cm). 57CoCl2, 111InCl3, 90YCl3, 88YCl , 64CuCl , and 67CuCl were obtained from ICN 3 2 2 (Costa Mesa, CA), Amersham (Arlington Heights, IL), NEN (Boston, MA), Los Alamos National Laboratory (Los Alamos, NM), Washington University (St. Louis, MO), and Brookhaven National Laboratory (Upton, NY), respectively. Ultrapure water (18 MΩ cm) was used for all procedures, and all other reagents were of the highest purity obtainable. Thin-Layer Chromatography. Thin-layer chromatography was performed on ITLC plates (silica gel 60F254, 0.5 × 10 cm, EM Science, Gibbstown, NJ), using 10% (w/v) ammonium acetate:methanol (1:1) as the mobile phase. In this system, radiometal-labeled antibody remains at the origin, while radiometal-DTPA complexes migrate to Rf ) 0.7-0.9. Radiation counting of TLC plates

10.1021/bc0000886 CCC: $20.00 © 2001 American Chemical Society Published on Web 03/06/2001

Technical Notes

was performed with a Packard Cobra Auto-Gamma model 5003 counter with the appropriate energy window set for 57Co or 111In. High-Performance Liquid Chromatography. HPLC was performed at room temperature on a Gilson chromatograph (models 307 pump, 506C System Interface Module, 715 System Controller Software, and FC 203 Fraction Collector). The column, solvent system, and flow rate used are described below. UV detection was accomplished at 280 nm using a Gilson 112 detector (10mm, 11-µL analytical flow cell). Radioactivity detection was accomplished using a NaI detector and a Technical Associates PRS-5 analyzing miniscaler/ratemeter. Ultraviolet Spectrophotometry. Concentrations of antibody were determined by UV spectrophotometry, measuring the absorbance at 280 nm (A280 at 1 mg/mL ) 1.42, within 5% of the concentration determined by amino acid analysis). UV measurements were obtained on a Pharmacia LKB Ultrospec III spectrophotometer, using a 1-cm sample cell. Preparation of N-Hydroxysulfosuccinimidyl DOTA (1). A solution of 16.2 mg of DOTA in 810 µL of H2O was adjusted to pH 5.45 with 80 µL of 1 M NaOH and cooled to 4 °C. To 239 µL (4.35 mg, 10.7 µmol) of the DOTA solution was added 2.33 mg (10.7 µmol) of sulfoNHS in 117 µL of H2O, freshly prepared at 4 °C. Then 8.25 µL (0.207 mg, 1.08 µmol) of EDC, freshly prepared in H2O (25 mg/mL), was added, and the reaction mixture was stirred at 4 °C for 30 min. The theoretical concentration of 1 in the reaction mixture was 2.97 mM. Prior to being added to the mAb, the pH of the reaction mixture was adjusted to 7.3 with 40 µL of 0.2 M Na2HPO4, pH 9.2. Preparation of cT84.66-DOTA Conjugate. An aliquot of 8.64 mg of cT84.66 in 1 mL of PBS, pH 7.4, was dialyzed against 1 L of 0.1 M Na2HPO4, pH 7.5, containing approximately 1.2 g of Chelex 100, for 42 h at 4 °C, with one buffer change. Then, the solution of 1 (1.08 µmol of DOTA-OSSu theoretical) was added to 8.06 mg (53.7 nmol) of cT84.66 in 1 mL of 0.1 M Na2HPO4, pH 7.5. The reaction mixture was incubated at 4 °C for 24 h with continuous end-over-end mixing, after which it was dialyzed against 1 L of 10 mM Na2HPO4/150 mM NaCl, pH 7.5, containing approximately 1.2 g of Chelex 100, for 24 h at 4 °C. The conjugate was then dialyzed against 1 L of 0.25 M ammonium acetate, pH 7.0, containing approximately 1.2 g of Chelex 100, for 139 h at 4 °C, with five buffer changes. Determination of the Average Number of Chelates per Antibody Molecule. An aliquot of 10 µL of cT84.66-DOTA, containing approximately 400 pmol of conjugate, was added to 10 µL of 0.25 M ammonium acetate, pH 7.0, followed by 10 µL of a standardized CoCl2 solution (402 µM, 4.02 nmol, containing >350000 cpm/ µL of 57CoCl2) or InCl3 solution (428 µM, 4.28 nmol, containing >4.40 × 106 cpm/µL of 111InCl3). The reaction mixture was incubated at 43 °C for 3 h, after which 3.3 µL (33 nmol) of 10 mM DTPA, pH 6.0, was added. The reaction mixture was incubated at room temperature for 15 min. An aliquot of 1 µL of the reaction mixture was spotted onto an ITLC plate, which was developed until the solvent front had migrated 7 cm from the origin. The plate was cut in 1-cm increments from 1 cm above the origin to 6 cm above the origin, and the resulting seven sections were counted in the gamma counter. The number of chelates per antibody molecule was calculated from the ratio of counts remaining at the origin (0-1 cm, Rf ) 0-0.14) to the total number of counts, using the method of Meares et al. (17).

Bioconjugate Chem., Vol. 12, No. 2, 2001 321

Radiolabeling of cT84.66-DOTA. Representative conditions are given for labeling cT84.66-DOTA with the radiometals 111In(III), 88,90Y(III), and 64,67Cu(II). In the 111In and 90Y labeling reactions, 88.8 µL of 0.25 M ammonium acetate, pH 7.0, was added to 2.0 mCi of 111InCl3 in 111 µL of 0.04 N HCl or 6.0 mCi of 90YCl3 in 4.6 µL of 0.05 N HCl, followed by 0.250 mg of cT84.66-DOTA in 50.4 µL of 0.25 M ammonium acetate, pH 7.0. The reaction mixture was incubated at 43 °C for 1 h, and then the reaction mixture was made 1 mM in DTPA with the addition of one-ninth of a reaction volume of 10 mM DTPA, pH 6.0. The reaction mixture was allowed to stand at room temperature for 15 min, after which the 111Inor 90Y-labeled antibody was purified by size-exclusion HPLC using a TosoHaas TSKgel G2000 SW column (7.5 × 300 mm, 10 µm), an isocratic mobile phase of normal saline, and a flow rate of 1.0 mL/min. The mAb peak, eluting at 10.3 min retention time, was collected in 0.5mL fractions containing 1 drop of 25% (w/v) human serum albumin (HSA) each. For 88Y labeling, a longer reaction time was needed to achieve optimal labeling efficiency, presumably because of higher concentrations of trace metal contaminants in preparations of this isotope. In a typical 88Y labeling reaction, 190 µL of 0.25 M ammonium acetate, pH 7.0, was added to 0.287 mCi of 88YCl3 in 102 µL of 0.1 N HCl, and then 0.300 mg of cT84.66-DOTA in 69 µL of 0.25 M ammonium acetate, pH 7.0, was added. The reaction mixture was incubated at 43 °C for 3.5 h, after which 40.1 µL of 10 mM DTPA, pH 6.0, was added. The reaction mixture was incubated at 37 °C for 15 min, and the 88Ylabeled conjugate was purified by size-exclusion HPLC in the same manner as the 111In-labeled antibody. In the 64Cu labeling reaction, 120 µL of 0.1 M ammonium citrate, pH 5.5, was added to 1.4 mCi of 64CuCl2 in 3.5 µL of 0.1 N HCl, followed by 0.205 mg of cT84.66DOTA in 45 µL of 0.25 M ammonium acetate, pH 7.0. For 67Cu labeling, 200 µL of 0.1 M ammonium citrate, pH 5.5, was added to 1.17 mCi of 67CuCl2 in 100 µL of 0.1 N HCl, and then 0.228 mg of cT84.66-DOTA in 50 µL of 0.25 M ammonium acetate, pH 7.0, was added. The reaction mixtures were incubated at 43 C for 1 h, after which they were made 1 mM in EDTA with the addition of one-ninth of a reaction volume of 10 mM EDTA, pH 5.5. The reaction mixtures were allowed to stand at room temperature for 15 min, and the 64Cu- or 67Cu-labeled mAb was purified by size exclusion HPLC in the same manner as the 111In-labeled conjugate. Immunoreactivity Determination. A dilution of the purified radiolabeled cT84.66-DOTA conjugate, containing approximately 200 000 cpm of radioactivity, was mixed with a 20-fold molar excess of purified CEA (18, 19) in 150 µL of 1% HSA/PBS, pH 7.4. A control sample was prepared in an identical manner, except that CEA was not added. The reaction mixtures were incubated at 37 °C for 15 min with continuous end-over-end mixing, after which aliquots of 100 µL were analyzed by gel filtration HPLC, using two Pharmacia Superose 6 HR 10/30 columns (1 × 30 cm) in series and an isocratic mobile phase of 0.05 M Na2SO4/0.02 M NaH2PO4/0.05% NaN3, pH 6.8, at a flow rate of 0.5 mL/min. Immunoreactivity was calculated as the percentage of the total radioactivity shifted to complexes with apparent molecular weights higher than that of the mAb. Isoelectric Focusing. Aliquots of 5 µg of cT84.66 and each of three lots of cT84.66-DOTA were analyzed by isoelectric focusing (IEF) on pH 3-10 precast gels (NOVEX, Invitrogen Corp., Carlsbad, CA). IEF gels were run at constant voltage for 1 h at 100 V, 1 h at 200 V,

322 Bioconjugate Chem., Vol. 12, No. 2, 2001

Lewis et al.

Scheme 1

Figure 1. Average number of chelates per molecule of human IgG conjugated with 100 theoretical equiv of DOTA-OSSu, determined by a 57Co(II) binding assay, as a function of the pH at which DOTA-OSSu was prepared.

and then 30 min at 500 V. The gels were fixed in 0.14 M 5-sulfosalicylic acid/0.7 M trichloroacetic acid for 30 min at room temperature, stained with Coomassie Brilliant Blue R-250 for 30 min at room temperature, and destained in H2O overnight. RESULTS AND DISCUSSION

The previously reported method for conjugation of monoclonal antibodies with N-hydroxysulfosuccinimidyl DOTA (15) was a simple, aqueous phase procedure using only commercially available reagents (Scheme 1). However, large-scale preparation of clinical lots of mAbDOTA conjugates, using 250 mg of antibody, would consume approximately 675 mg of DOTA. Therefore, it was desirable to improve the efficiency of the conjugation reaction, to reduce the amount of chelating agent required. Several modifications to the original procedure were made. First, the pH of the active ester synthesis was optimized. In the original method, activation of trisodium DOTA with EDC and sulfo-NHS was performed at ambient pH, approximately 2.5-2.6. However, watersoluble carbodiimides are more stable at higher pH, in the range of 4 to 10 (20, 21). Above pH 7, the hydrolytic stability of sulfo-NHS esters decreases markedly (22), and many active ester-mediated protein coupling reactions have been performed in the range of pH 5-6 (23, 24). Therefore, in the present work, human IgG was used as a model system and conjugated with 100 theoretical equiv of DOTA-OSSu prepared at pH 2.5, 3.75, 4.5, and 5.5. As shown in Figure 1, a linear relationship was found between the pH of activation and the degree of antibody modification. Determination of the average number of chelates per antibody molecule indicated that the efficiency of conjugation increased approximately 6-fold when DOTA-OSSu was synthesized at pH 5.5, compared to ambient pH. Second, pH and buffer conditions of the antibody conjugation reaction were improved. Several groups have

reported optimum coupling yields at pH values of 5-8 (21-24), where rates of amidation of succinimidyl esters increase more rapidly than rates of hydrolysis. In the previously described method (15), the anti-CEA mAb cT84.66 was dialyzed against 0.25 M ammonium acetate prior to dialysis against the conjugation buffer, 0.1 M NaHCO3/0.1 M K2HPO4, pH 8.5. Under these conditions, the concentration of ammonium acetate in the conjugation reaction was 0.41 mM, resulting in a theoretical DOTA-OSSu:ammonia molar ratio of 7.4:1. Even though ammonia is a relatively poor nucleophile in aqueous media, competing aminolysis of the DOTA active ester may have decreased the efficiency of antibody conjugation. In the present work, no ammonia-containing buffers were used for dialysis prior to conjugation, and cT84.66 was reacted with DOTA-OSSu in sodium phosphate buffer at pH 7.5. On the basis of previously published studies, it was anticipated that the lower pH conditions would decrease the rate of hydrolysis of N-hydroxysulfosuccinimidyl DOTA by a factor of 2-2.5 (22) while maintaining the reactivity of the active ester with primary amines at neutral to slightly alkaline pH (24, 25). EDC has been shown to react readily with inorganic phosphate to form an O-phosphoisourea, which hydrolyzes rapidly to give a substituted urea and phosphate (20). Thus, the pH of the DOTA-OSSu solution was adjusted to 7.0-7.5 with sodium phosphate before being added to the mAb. This procedure not only minimized the potential for mAb cross-linking by unreacted EDC, but also reduced the acidity of the active ester preparation and the possibility of protein denaturation. When analyzed by gel filtration HPLC, antibody-DOTA conjugates prepared by the previously published procedure were found to contain between 2.35 and 6.00% antibody dimers. Conjugates prepared under the improved pH and buffer conditions contained 0 to 0.862% dimers, as determined by gel filtration HPLC. Model conjugation studies with cytochrome c (15) suggested that the reaction was equally efficient at 4 °C and room temperature. However, cytochrome c is a small, hydrophilic, and very lysine-rich protein and, compared to antibodies, may react with active esters at a rate more comparable to that of competing hydrolysis. Hydrolysis of N-hydroxysulfosuccinimide esters is an order of magnitude slower at 0 °C than at 31 °C, increasing the halflife of active esters from 0.87 h at 31 °C to 11 h at 0 °C (22). Thus, mAb conjugations with N-hydroxysulfosuccinimidyl DOTA were performed at 4 °C to minimize the rate of hydrolysis of the active ester. It was expected that a long incubation time (24 h) at low temperature (4 °C) would maximize both the lifetime of DOTA-OSSu in solution and the efficiency of antibody conjugation. Using the optimized activation and conjugation conditions, the reaction of N-hydroxysulfosuccinimidyl DOTA with antibodies was approximately 6-fold more efficient than when the previously published procedure was used (Figure 1). Therefore, for subsequent conjugations the molar ratio of DOTA-OSSu:cT84.66 was reduced from

Technical Notes

Bioconjugate Chem., Vol. 12, No. 2, 2001 323

Table 1. DOTA Activation and cT84.66 Conjugation Conditions lot no.

molar ratio DOTA:sulfo-NHS:EDC:mAb

activation pH

conjugation buffer

chelates/mAb

RJ121296

1000:1000:100:1

2.55

1.7

010297NC 021997 MA071098

200:200:20:1 200:200:20:1 200:200:20:1

5.18 5.15 5.45

0.1 M NaHCO3/0.1 M K2HPO4, pH 8.5 0.1 M Na2HPO4, pH 7.5 0.1 M Na2HPO4, pH 7.5 0.1 M Na2HPO4, pH 7.5

3.1 3.1 3.8

Table 2. Radiometal Labeling of cT84.66-DOTA radiometal

n

labeling ratios (mCi/mg)

labeling efficiency (%)

specific activities (mCi/mg)

immunoreactivity (%)

111Inb

19 14 2 1 1

0.723-38.1 0.185-24.2 0.957-1.00 5.22 6.83

98.9 ( 1.0 86.3 ( 9.9 94.1 ( 2.2 61.0 100.0

0.718-37.7 0.181-19.4 0.915-0.925 3.18 6.83

99.9 ( 0.3 98.6 ( 2.9 NDa ND 100.0

90Yb 88Y 67Cu 64Cu a

ND ) not determined. b Reaction of unconjugated, demetalated cT84.66 with

100:1 to 20:1. Table 1 gives the activation and conjugation conditions, as well as the number of chelates per mAb determined by 57Co and 111In binding assays, for one lot of cT84.66-DOTA prepared by the original method and three lots of cT84.66-DOTA prepared using the procedure described here. The results of the radiometal binding assays show that conjugates prepared by the two procedures contain similar numbers of functional chelates, yet the quantities of reagents used to conjugate the antibody were reduced by a factor of 5 when the new method was used. DOTA conjugates of cT84.66, prepared by the original procedure and the improved method, were analyzed by isoelectric focusing (Figure 2). The pI of unmodified cT84.66 (lane 1) was determined to be 6.5-7.3 by IEF. Modification with DOTA resulted in expected decreases in the isoelectric points of the mAb conjugates. The pI of cT84.66-DOTA, conjugated by the previously published method (lot RJ121296, lane 5), was found to be 5.0-6.7. However, a pair of predominant bands at a pI value of 5.3 suggested the presence of a relatively large population of highly modified mAb species. In contrast, cT84.66DOTA conjugates prepared by the new method (lots MA071098, 010297NC, and 021997; lanes 2, 3, and 4) showed a more uniform distribution of antibody modification over a pI range of 5.2-7.0. Thus, the improved conjugation procedure described here gives a more homogeneous product, with fewer highly modified conjugates. Overmodification of mAbs with BCAs can lead to decreases in immunoreactivity and tumor uptake (26), so it was desirable to reduce the fraction of heavily conjugated species. The results of 111In, 90Y, 88Y, 64Cu, and 67Cu labeling reactions of cT84.66-DOTA under no-carrier-added conditions are listed in Table 2. The 111In and 90Y labeling reactions were performed over a wide range of specific activities, for the purposes of animal biodistribution, patient imaging, patient therapy, and human antichimeric antibody (HACA) studies. When unconjugated, demetalated cT84.66 was incubated with 111In or 90Y, no nonspecific binding of either radiometal to the mAb was observed after DTPA challenge and HPLC purification. Labeling of cT84.66-DOTA proceeded with >85% efficiency with all radiometals except 67Cu. The relatively low specific activity of 67Cu (185 Ci/mmol, compared to 20 000 Ci/mmol for 64Cu) likely contributed to its lower labeling efficiency, because of competition by cold copper for chelation sites on the conjugate. Compared to the previously published work (15), 111In labeling was increased by 15%, and 90Y labeling was improved by 27%.

111In

or

90Y

resulted in 0% radiometal incorporation.

Figure 2. IEF analysis of unconjugated cT84.66 (lane 1) and cT84.66-DOTA conjugates prepared by the method reported herein, Lot MA071098 (lane 2), lot 010297NC (lane 3), lot 021997 (lane 4), and cT84.66-DOTA prepared by the previously published method (15), lot RJ121296 (lane 5). Isoelectric point markers are shown to the right.

However, the standard deviation of 90Y labeling efficiency was larger than those of the other radiometals. The variation in 90Y labeling did not correlate with specific activity, mAb conjugate lot, or storage of the conjugate over a period of 14 months. Rather, the efficiency of labeling seemed to vary with the lot of 90Y used. Table 2 also lists the immunoreactivities of the radiometallabeled conjugates, as determined by a solution-phase CEA binding assay. For all conjugates analyzed, immunoreactivity was essentially quantitative. By optimizing pH, buffer, and temperature conditions for both the preparation of N-hydroxysulfosuccinimidyl DOTA and its conjugation to the anti-CEA monoclonal antibody cT84.66, several improvements to the previously published procedure were made. These improvements

324 Bioconjugate Chem., Vol. 12, No. 2, 2001

included a 6-fold increase in conjugation efficiency, a 3-7fold decrease in antibody dimerization, a more homogeneous population of conjugate species, and a 5-fold reduction in the quantities of DOTA and coupling reagents required for the conjugation. Furthermore, 111In and 90Y labeling of cT84.66-DOTA was increased significantly, and high specific activity labeling with Cu radioisotopes was achieved, while maintaining quantitative immunoreactivity. This procedure has been scaled up to produce two lots of 233 and 215 mg, respectively, of clinical-grade cT84.66-DOTA. We are currently evaluating 90Y-labeled cT84.66-DOTA in a Phase I/II clinical trial for radioimmunotherapy of CEA-positive malignancies. ACKNOWLEDGMENT

The authors wish to thank Randall Woo and Militza Bocic for excellent technical assistance, as well as Michael J. Welch, Deborah W. McCarthy, and Todd A. Perkins of Washington University in St. Louis for providing 64Cu. This work was supported by Research Grant CA43904 from the National Cancer Institute, NIH. LITERATURE CITED (1) Waldmann, T. A. (1991) Monoclonal Antibodies in Diagnosis and Therapy. Science 252, 1657-1662. (2) Schubiger, P. A., Alberto, R., and Smith, A. (1996) Vehicles, Chelators, and Radionuclides: Choosing the “Building Blocks” of an Effective Therapeutic Radioimmunoconjugate. Bioconjugate Chem. 7, 165-179. (3) Meares, C. F. (1986) Chelating Agents for the Binding of Metal Ions to Antibodies. Nucl. Med. Biol. 13, 311-318. (4) Gansow, O. A. (1991) Newer Approaches to the Radiolabeling of Monoclonal Antibodies by Use of Metal Chelates. Nucl. Med. Biol. 18, 369-381. (5) Liu, Y., and Wu, C. (1991) Radiolabeling of Monoclonal Antibodies with Metal Chelates. Pure Appl. Chem. 63, 427-463. (6) Kozak, R. W., Raubitschek, A., Mirzadeh, S., Brechbiel, M. W., Junghaus, R., Gansow, O. A., and Waldmann, T. A. (1989) Nature of the Bifunctional Chelating Agent Used for Radioimmunotherapy with Yttrium-90 Monoclonal Antibodies: Critical Factors in Determining in Vivo Survival and Organ Toxicity. Cancer Res. 49, 2639-2644. (7) Loncin, M. F., Desreux, J. F., and Merciny, E. (1986) Coordination of Lanthanides by Two Polyamino Polycarboxylic Macrocycles: Formation of Highly Stable Lanthanide Complexes. Inorg. Chem. 25, 2646-2648. (8) Cacheris, W. P., Nickle, S. K., and Sherry, A. D. (1987) Thermodynamic Study of Lanthanide Complexes of 1,4,7Triazacyclononane-N,N′,N′′-triacetic Acid and 1,4,7,10-Tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic Acid. Inorg. Chem. 26, 958-960. (9) Kasprzyk, S. P., and Wilkins, R. G. (1982) Kinetics of Interaction of Metal Ions with Two Tetraaza Tetraacetate Macrocycles. Inorg. Chem. 21, 3349-3352. (10) Wang, X., Jin, T., Comblin, V., Lopez-Mut, A., Merciny, E., and Desreux, J. F. (1992) A Kinetic Investigation of the Lanthanide DOTA Chelates. Stability and Rates of Formation and of Dissociation of a Macrocyclic Gadolinium(III) Polyaza Polycarboxylic MRI Contrast Agent. Inorg. Chem. 31, 10951099. (11) Deshpande, S. V., DeNardo, S. J., Kukis, D. L., Moi, M. K., McCall, M. J., DeNardo, G. L., and Meares, C. F. (1990) Yttrium-90-Labeled Monoclonal Antibody for Therapy: Labeling by a New Macrocyclic Bifunctional Chelating Agent. J. Nucl. Med. 31, 473-479.

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