Bioconjugate Chem. 1990, 1, 269-273
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Protein Radiohalogenation: Observations on the Design of N-Succinimidyl Ester Acylation Agents Ganesan Vaidyanathan and Michael R. Zalutsky' Duke University Medical Center, Department of Radiology, Box 3808, Durham, North Carolina 27710. Received April 11, 1990 In previous studies we have demonstrated that antibodies radioiodinated with N-succinimidyl 3-iodobenzoate (SIB) are less susceptible to loss of radioiodine in vivo than antibodies iodinated directly by electrophilic substitution on their tyrosine residues with Iodogen. Since the Bolton-Hunter reagent, N-succinimidyl3-(4-hydroxy-3-iodophenyl)propionate, is identical with SIB except that it contains a hydroxyl group on the aromatic ring and a two-methylenespacer, a comparisonof their coupling chemistry and in vivo behavior was performed to better understand the structural requirements for a useful iodinated acylation agent. Protein concentration and pH had a significant effect on the coupling efficiency of both SIB and the Bolton-Hunter reagent; however, protein-labeling yields with SIB were generally higher by a factor of 2. Paired-label biodistribution studies in mice demonstrated that thyroid uptake (a monitor of dehalogenation)of antibody labeled by the Bolton-Hunter method was twice that of antibody labeled with SIB but only 7 r0 of that observed for antibody labeled with Iodogen. These results suggest that even minor differences in iodination site can profoundly alter the retention of label on a protein in vivo.
INTRODUCTION Radioiodination of monoclonal antibodies (MAbs)' is a labeling approach which offers certain advantages over the use of metallic nuclides such as lllIn (1). Because of the availability of multiple y-emitting iodine nuclides, direct comparison of different MAbs, labeling methods, or routes of injection is possible by paired-label analyses, greatly facilitating the investigation of the basic processes influencing MAb distribution. In addition, the nuclear properties of 1231 are nearly ideal for either planar imaging or single-photon emission tomography (2). Conventionalmethods for the radioiodination of proteins such as the Iodogen method (3) involve direct electrophilic substitution of the iodine ortho to the hydroxyl group on tyrosine residues ( 4 ) . MAbs labeled by this approach undergo rapid deiodination after administration in vivo (41, a factor which is a major impediment to the utilization of radiohalogenated MAbs. In an attempt to develop improved methods for protein radioiodination, our laboratory has been investigating the influence of the chemical nature of the MAb iodination site on subsequent behavior in vivo. Because of the better biologic properties generally associated with proteins labeled by the Bolton-Hunter method (5) (presumably because of the nonoxidative conditions which are employed), this reagent, N-succin(BH, imidyl 3-(4-hydroxy-3-[~25I]iodophenyl)propionate Chart I) was utilized as the point of departure for the design of other protein iodination reagents. For example, we have developed a conceptually similar compound, N-succinimidyl3-iodobenzoate(SIB) (Chart I), which is obtained by the iododestannylation of N-succinimidyl 3- (tri-n-butylstanny1)benzoate (ATE) (Chart I ) ( 6 ) . Subsequent reports by other investigators have described 1 Abbreviations used: MAbs, monoclonal antibodies; BH, N-succinimidyl3-(4-hydroxy-3[ 125I]iodophenyl)propionate;SIB N-succinimidyl 3-iodobenzoate; ATE, N-succinimidyl 3-(tri-nbutylstannyl) benzoate.
Chart I. Structures of ATE, SIB, and Bolton-Hunter Reagent h
0 X Y BH: X = I, Y = OH, n = 2 ATE: X = SnBu3, Y = H, n = 0 SIB: X = I , Y = H , n = O
Chart 11. Iodination Site on Protein
[-HNCHC-]0ii I
I Y BH: X = -(CHZ)~NHCO(CH~)*)-, Y = OH SIB: X = -(CH&NHCO-, Y = H Iodogen: X = -CHz-, Y = OH
the use of N-succinimidyl 4-iodobenzoate for labeling proteins (7,8). As illustrated in Chart 11, labeling proteins using both BH and SIB results in modification of lysine residues on the protein. However, two structural differences were incorporated into the design of SIB in an attempt to make this reagent more useful for in vivo applications (Chart I). First, the two-carbon spacer between the aromatic ring and the activated ester was removed in order to increase protein coupling yields by minimizing competitive hydrolysis. And second, unlike BH, SIB lacks the phenolic hydroxyl group ortho to the iodine atom. It was speculated that the extensive deiodination of proteins which is observed in vivo could be minimized by decreasing the structural similarity of the protein iodination site to thyroid hormones, for which multiple dehalogenases are known to exist (9-1 I).
1043-1802/90/2901-0269$02.50/00 1990 American Chemical Society
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Several paired-label studies have documented the fact that intact MAbs and their F(ab')2 fragments labeled by using the ATE method are more inert to dehalogenation than those labeled by direct electrophilic substitution on tyrosine residues (Iodogen method) (12, 13). These experiments facilitated the comparison of our results to those reported in the literature for directly labeled MAbs; however, testing the original design hypothesis for SIB requires a direct comparison to MAbs labeled with BH. In this study, we have compared the protein-coupling chemistries of SIB and BH. In addition, data will be presented which suggests that presence or absence of a hydroxyl group ortho t o the site of iodination is not necessarily the primary factor influencing the loss of radioiodine from MAbs in vivo. EXPERIMENTAL PROCEDURES
Reagents. ATE was synthesized and purified by using previously published procedures (6). The mono[ 1 2 q iodinated form of the Bolton-Hunter reagent was obtained from Amersham Corp. (Arlington Heights, IL) and sodium [131I]iodide was purchased from Du Pont-New England Nuclear (Boston, MA). Goat immunoglobulin (IgG) was purchased from Sigma Chemical Co. (St. Louis, MO). MAb 81C6 is of the IgG2b isotype and reacts with an epitope of the extracellular matrix antigen tenascin (14). It was obtained as a gift from Dr. Dare11 Bigner, Department of Pathology, Duke University Medical Center. S y n t h e s i s of N - S u c c i n i m i d y l 3-[1311]Iodobenzoate (SIB). Radioiodination of ATE was accomplished as described in an earlier publication (15). Briefly, to 1-2 pL of 1311in 0.1 N NaOH in a glass, conical vial was added twice the volume of 3 9" acetic acid in CHCl3 followed by 15 pL of tert-butyl hydroperoxide (10% in CHC13) and 5 pL of ATE (0.5pmol in CHC13). After stirring for 30 min at room temperature, [1311]SIBwas isolated by HPLC. The separation system consisted of an Alltech silica gel column eluted with hexane/ethyl acetatelacetic acid (70:29.88: 0.12). General Method for Labeling IgG Using SIB and BH. The standard labeling conditions used were as follows: For the ATE method, the HPLC fractions containing [1311]SIBwere concentrated to about 50-100 pL and transferred, with the aid of a small volume of ethyl acetate, t o a 0.5-dram glass vial. T h e solvent was evaporated with a gentle stream of argon. In the case of BH, an appropriate volume of the benzene/DMF solution containing the 1251-labeledactive ester was transferred to a 0.5-dram vial and evaporated with a gentle stream of argon. Goat IgG or 81C6 MAb (75 pL, 150 pg) in 0.1 M borate buffer, pH 8.5, was added to the vial containing either [13lI]SIB or [12SI]BH,and the mixture was incubated on ice a t 4 "C for a period of 20 min with gentle shaking. The reaction was terminated by the addition of 300 p L of 0.2 M glycine in 0.1 M borate. The radioiodinated protein was isolated from lower molecular weight impurities with a Sephadex G-25 column. Protein-associated activity, determined by precipitation with 20% trichloroacetic acid, was greater than 95% for all preparations. Labeling 81C6 IgG with 1311Using Iodogen. For use in some of the biodistribution studies, 81C6 MAb was labeled with 1311by using a variation of the original Iodogen method (3). MAb 81C6 (200 pg in 220 pL 100 mM phosphate buffer, pH 7.4) was added to sodium [1311]iodide in a glass vial coated with 10 pg of Iodogen (Pierce Chemical Co., Rockford, IL). After a 10-min reaction a t room temperature, radioiodinated 81C6 was purified by
chromatography over a Sephadex G-25 column. The trichloroacetic acid precipitibility of this preparation was 99%. Effect of pH. Goat IgG a t a concentration of 2 mg/ mL was prepared in 0.1 M borate buffers with pH in the range 8.5-10.0. To both [1311]SIBand [12SI]BHwas added 75 pL of the various protein solutions. Protein coupling efficiency was calculated by dividing the activity eluting in the void volume of the Sephadex G-25 column by the total activity added to the column. Activity levels were measured with a Capintec CRC-7 dose calibrator. Two to five determinations were performed a t each pH. Effect of Protein Concentration. Goat IgG in pH 8.5, 0.1 M borate buffer was prepared a t concentrations of 1, 2, 3, 4,and 10 mg/mL. To both [1311]SIB and [1251]BH was added 75 pL of each goat IgG solution. Methods employed for separating t h e labeled protein a n d determining coupling efficiency were as described above. Two to five determinations were performed a t each protein concentration. Biodistribution Studies. MAb 81C6 was labeled with 1251 by using BH and with 1311by using SIB as described above. Specific activities for the preparations, determined by measuring the protein concentration spectrophotometrically and the radioactivity level with the dose calibrator, were approximately 1 pCi/pg. BALB/c mice weighing 20-25 g were injected in the tail vein with 3 pg each of 1251-and 1311-labeled81C6. Groups of 5 or 6 mice were sacrificed by ether overdose a t 3 , 4 , 5 , 6 , and 7 days after injection for paired-label biodistribution analysis. This protocol was repeated twice. An additional set of mice was by using injected with 2 pg each of 81C6 labeled with 1251 BH and with 1311 by using Iodogen. Animals were dissected; tissues of interest were removed, weighed, washed with saline, and counted for both 1251 and 1311 activity with an LKB Model 1282 dual-channel y-counter. Counting data were corrected for crossover of 1311activity into the 1251counting window. The percent injected dose in each tissue for both nuclides was calculated by comparison to injection standards of appropriate count rate. Blood was assumed to represent 6% of total body weight. Statistical Analysis. Comparisons between t h e coupling efficiencies of SIB and BH were made by using the Student's t test (16). Since the tissue distribution experiments were performed in paired-label format, a direct comparison of the different iodination methods, with each animal serving as its own control, was possible. Data were analyzed by using a paired t test (16). In both analyses, only p < 0.05 has been considered to be statistically significant. RESULTS AND DISCUSSION
Proteins can be radioiodinated either directly by electrophilic substitution on tyrosine residues or by conjugation of a labeled compound to the eamino group of lysines, thus permitting the labeling of protides lacking a reactive tyrosine residue. An additional advantage is that conjugation radioiodination methods avoid exposing the protein to oxidants. In studies comparing the biologic activity of several proteins and peptides labeled directly and by using the Bolton-Hunter method, use of the later yielded greater retention of immunological activity (5, 17). Since preservation of immunocompetence after radiolabeling is a critical factor in the labeling of MAbs, acylation agents of this type might be useful for radioimmunoscintigraphic and therapeutic applications. In an attempt to create a Bolton-Hunter analogue that would be more suitable for routine use in vivo, a method
Bioconjugate Chem., Voi. 1, No. 4, 1990 271
Protein Radiohalogenation
a
80
I
ATE
BH
e-. 0-0
1
I
1
i
4
a
I
20
" ,
0
2
4
8
8
10
12
Goat IgG (mg/mL)
Figure 1. Effect of protein concentration on protein-labeling efficiency for the ATE and Bolton-Hunter methods. Reaction conditions, pH = 8.5 for 20 min at 4 "C.
"
I
8.0
8.5
9.0
9:5
10.0
10.5
PH
Figure 2. Effect of pH on protein-labelingefficiency for the ATE and Bolton-Hunter methods. Reaction conditions,150 pg of goat IgG per 75 fiL, 20 min at 4 "C.
was developed for synthesizing N-succinimidyl 3-iOdObenzoate (SIB)from N-succinimidyl3-(tri-n-butylstannyl)benzoate via electrophilic destannylation (6). The purpose of the present study was to evaluate by direct comparison to the Bolton-Hunter reagent two hypotheses used in the design of SIB. Coupling Chemistry. A major difficulty with the Bolton-Hunter reagent and other conjugation labeling methods (18) is that conjugation efficiencies are low, generally on the order of 15-30% (18,19). We speculated that omitting the two-carbon spacer between the aromatic ring and the N-succinimidyl ester moiety should increase conjugation efficiency to the protein by minimizing loss of labeled ester as a consequence of hydrolysis. In Figure 1, the coupling efficiency for BH and SIB at pH 8.5 and a reaction time of 20 min is compared. At 1 mg/mL goat IgG in a 75 FL reaction volume, the yield with SIB was 30.3 f 5.4%, a value 1.25times that obtained with the BH reagent (24.3 f 4.295, difference not significant, p = 0.06). With both BH and SIB,coupling yield increased with increasing protein concentration until 3 mg/mL, after which a plateau was reached. In both cases, the results do not appear to represent first-order dependence of the reaction rate on protein concentration. It is important to note that, a t all concentrations above 1 mg/mL, the coupling yields obtained with SIB were 1.8-2.3 times greater than those observed with BH ( p < 0.005). Over the pH range 8.5-10.0, a t a fixed protein concentration of 2 mg/mL, maximum yields were obtained for both BH and SIB at a pH of 9.5 (Figure 2). It seems likely that the increased coupling efficiency at higher pH is a consequence of greater deprotonation of lysine €-amino groups at higher pH, resulting in more amine sites available for reaction with the N-succinimidyl esters. Lower yields at pH 10 could be the result of competitive hydrolysis and/ or partial precipitation of goat IgG at this pH. Again,
conjugation efficiency for SIB was about twice that of BH at all pH's (38.3 f 0.4% BH; 82.0 f 4.9% SIB at pH 9.5; p < 0.005). Yields obtained for coupling of SIB to goat IgG are in excellent agreement with our previous studies using goat IgG (6) and 81C6 MAb (13, 15) and are similar to those extrapolated from those of Wilbur et al. (7) and Khawli and Kassis (8)using slightly different reaction conditions. Likewise, coupling yields for BH are in qualitative agreement with those reported previously (5,19). However, since coupling efficiencies have been shown to be dependent on pH, protein concentration, and even the nature of the protein (19),the current study was performed to compare the utility of BH and SIB for the radioiodination of a protein under identical reaction conditions. The results of these experiments indicate that modifying the structure of the Bolton-Hunter reagent increased protein labeling yields by a factor of 2. It is presumed that the deletion of the two-carbon spacer between the N-succinimidyl ester and the aromatic ring resulted in a greater availability of active ester for amide-bond formation due to a decreased rate of ester hydrolysis. Indeed, when the Bolton-Hunter and SIB esters were exposed to PBS in the absence of protein, TLC analysis indicated that conversion to the corresponding acid was twice as fast for the BoltonHunter reagent. These results are in agreement with previous studies which have shown that the rate of hydrolysis of ethyl benzoates is considerably lower than that for ethyl esters of aryl compounds containing methylene spacers between t h e benzene ring and t h e ester functionality (20-22). Loss of Label in Vivo. Since we have hypothesized that the dehalogenation of conventionally radioiodinated proteins in vivo is facilitated by the presence of a hydroxyl group ortho to the iodine on an aromatic ring, iodination using the ATE reagent is accomplished via destannylation in order to avoid this potential problem. The reasons for this speculation are that lack of a hydroxyl group ortho to the iodine (a) decreases the structural similarity to iodotyrosine and thyroxine, known substrates for multiple dehalogenases (9-11), and (b) increases the strength of the C-I bond (23). Paired-label biodistribution studies were performed in normal mice to compare the pharmacokinetics of 81C6 MAb labeled by using both the ATE and Bolton-Hunter methods. As summarized in Table I, the tissue distribution of radioiodine generally is quite similar for MAb labeled using the two methods. However, as shown in Figure 3, thyroid uptake of radioiodine from MAb labeled by using the Bolton-Hunter method was significantly higher (p < 0.01-0.001) than that observed in the same mice for 81C6 labeled by using the ATE method, suggesting more rapid dehalogenation of MAb labeled by using the BoltonHunter method. Although differential dehalogenation of MAb labeled using t h e two different acylation agents was not unexpected, the fact that the ATE method offered only a %fold advantage was somewhat surprising in light of our previous studies (13) comparing this same MAb radioiodinated with ATE and with Iodogen. These results demonstrated that the thyroid uptake for 81C6 labeled with Iodogen was 40-100-fold higher than MAb labeled with ATE. Wilbur et al. (7) have reported that neck uptake of radioiodine was 2-8-fold lower for NR-ML-95 MAb labeled with p-iodophenyl benzoate compared to chloramine-T; however, for a F(ab'h fragment at similar time points, 25-100-fold differences were seen. In order to confirm the unexpectedly low thyroid uptake observed for 81C6 labeled with BH, the tissue distribution
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Table I. Paired-Label Tissue Distribution of Radioiodine in Normal Mice Following Injection of 81C6 Labeled with [12EI]BH and with [1311]SIB % injected dose per organ tissue
3 days
6 days
7 days
3.44 f 0.32 0.21 f 0.02 1.94 f 0.77 0.28 f 0.07 1.22 f 0.10 0.30 f 0.08 1.59 f 0.15 0.89 f 0.17" 7.88 f 0.49 3.26 f 0.22 13.28 f 1.51" 0.11 f 0.02
4 days 5 days Labeled with SIB 1.98 f 0.46 1.81 f 0.22" 0.13 f 0.02 0.12 f 0.01 0.76 f 0.24 1.24 f 0.42 0.17 f 0.05 0.21 f 0.05 0.72 f 0.13 0.67 f 0.11 0.16 f 0.05 0.14 f 0.01 1.00 f 0.24 0.97 f 0.11 0.46 f 0.14' 0.45 f 0.04 5.63 f 1.01 4.63 f 0.63 3.04 f 0.69 2.61 f 0.69 7.78 f 0.65" 8.41 f 1.61a 0.06 f 0.02 0.06 f 0.01
liver spleen lung heart kidney stomach small intestine large intestine muscle bone blood brain
1.64 f 0.80 0.09 f 0.04 0.81 f 0.27 0.15 f 0.07 0.53 f 0.21 0.13 f 0.07 0.67 f 0.29 0.37 f 0.16 3.15 f 1.29 1.39 f 0.59 5.87 f 2.56' 0.05 f 0.02
1.52 f 0.37b 0.09 f 0.01 0.73 f 0.47 0.13 f 0.03 0.50 f 0.09 0.10 f 0.03 0.73 f 0.09 0.30 f 0.04 3.07 f 0.18 1.40 f 0.18 5.45 f 0.75O 0.05 f 0.02
liver spleen lung heart kidney stomach small intestine large intestine muscle bone blood brain
3.28 f 0.29 0.21 f 0.02 1.80 f 0.69 0.27 f 0.07 1.23 f 0.12 0.30 f 0.09 1.74 f 0.13 1.18 f 0.25 7.61 f 0.41 3.09 f 0.18 11.93 f 1.44 0.10 f 0.02
Labeled with BH 1.87 f 0.42 1.64 f 0.19 0.13 f 0.02 0.11 f 0.01 0.73 f 0.22 1.11 f 0.32 0.16 f 0.04 0.19 f 0.05 0.73 f 0.12 0.66 f 0.10 0.16 f 0.05 0.13 f 0.01 1.07 f 0.24 0.96 f 0.12 0.59 f 0.20 0.51 f 0.08 4.47 f 1.16 5.38 f 0.85 2.88 f 0.58 2.34 f 0.65 6.92 f 0.59 7.30 f 1.44 0.06 f 0.01 0.06 f 0.01
1.46 f 0.71 0.08 f 0.03 0.71 f 0.23 0.13 f 0.06 0.52 f 0.20 0.12 f 0.06 0.66 f 0.28 0.44 f 0.17 2.85 f 1.13 1.19 f 0.52 4.96 f 2.18 0.04 f 0.02
1.34 f 0.33 0.08 f 0.01 0.64 f 0.42 0.11 f 0.03 0.49 f 0.09 0.09 f 0.02 0.72 f 0.11 0.35 f 0.07 2.96 f 0.51 1.31 f 0.25 4.51 f 0.68 0.04 f 0.02
Significance of difference determined by two-sided paired t test: p < 0.01; * p
( p > 0.05)
p
0.54
1
Days
Figure 3. Comparison of the percent injected dose of radioiodine localized in the thyroids of normal mice following the injection of 8lC6 monoclonal antibody labeled with lZ5I and l 3 l I by using the Bolton-Hunter and ATE methods, respectively.
of 81C6 labeled by using the Bolton-Hunter and Iodogen methods were compared directly in normal mice 3 days after injection. As shown in Table 11, significant differences in radioiodine uptake were observed between the two nuclides in only thyroid and stomach, the two tissues normally reflecting free-iodideuptake (12). Thyroid uptake associated with MAb labeled with the BoltonHunter reagent was about 13 times lower than for MAb labeled with Iodogen. In comparing the results obtained in this study with those reported previously, it is important to bear in mind that many factors can influence the magnitude of thyroid accumulation. Although the thyroid uptake observed for 81C6 labeled with SIB are in good agreement with a previous study, also performed in normal mice ( 2 4 ) ,they are 2-3-fold higher than those observed in athymic mice for both 81C6 and a nonspecific MAb in tumor-bearing athymic mice (13). Similarly, the thyroid uptake observed in the current study 3 days after injection of 81C6 labeled with Iodogen (5.97 f 0.73 % ) was also higher than that seen (13) in athymic mice (4.14 f 0.67%), suggesting that differences may exist in the catabolism of labeled MAbs
< 0.05;
p
< 0.02; other tissues, no significant difference
Table 11. Paired-Label Tissue Distribution of Radioiodine in Normal Mice 3 Days following Injection of 81C6 Labeled with [12sI]BH and with [lSII]Iodogen % iniected dose per organ tissue BH Iodogen liver 2.81 f 0.50 2.88 f 0.60 spleen 0.19 f 0.03 0.19 f 0.03 lung 0.72 f 0.20 0.75 f 0.22 heart 0.22 f 0.04 0.23 f 0.04 kidney 1.05 f 0.06 1.15 f 0.06 0.46 f 0.05 stomach 0.22 f 0.05" small intestine 1.46 f 0.25 1.29 f 0.18 large intestine 0.95 f 0.15 0.87 f 0.21 thyroid 0.44 f 0.06" 5.97 f 0.73 muscle 7.42 f 2.38 6.84 f 1.04 bone 3.04 f 0.31 3.56 f 0.48 13.50 f 2.02 blood 12.50 f 1.78 0.09 f 0.01 brain 0.09 f 0.03 Significance of difference determined by two-sided paired t test: p < 0.001; other tissues, no significant difference (p > 0.05).
in normal versus athymic mice. In addition, Dumas et al. (25) has reported that the deiodinase activity of sheep liver microsomes in vitro for aryl iodides varied from lot to lot and was dependent on the nutritional and physiological state of the animal. Thus, paired-label protocols are essential to compare directly the catabolism of label from MAbs radioiodinated by using different methods. Although many antibodies and other proteins have been labeled by using the Bolton-Hunter method for in vitro applications, little data are available concerning their behavior in vivo. To our knowledge, no systematic study of MAbs labeled by using the Bolton-Hunter method has been reported which includes a determination of thyroid uptake. Although the pharmacokinetics of auromomycin would not be expected to be similar to those of an antibody, it is worth noting that Anatha Samy and coworkers (26) have reported that, 8 h after injection of this antibiotic labeled with 1251 by using the Bolton-Hunter method, uptake of lZ5I activity in the thyroid was only
Protein Radiohalogenation
0.13% of the injected dose. In addition, Schiff et al. (27) have reported that the radioactive catabolites formed following the cellular internalization of asialoglycoprotein labeled by the Bolton-Hunter and the iodine monochloride methods are different. While use of the ATE method for MAb radiohalogenation results in the lowest degree of thyroid accumulation, only a 2-fold advantage was observed relative to the Bolton-Hunter method in contrast to as much as a 100-fold advantage when compared to the Iodogen method. It thus appears that presence or absence of a hydroxyl group ortho to the iodination site is not the sole factor determining the thyroid uptake and presumably the dehalogenation of radioiodinated proteins. The specificity of deiodinases from sheep thyroidal and hepatic microsomes for 19 aryl iodides has been investigated by Dumas e t al. ( 2 5 ) . While 3 - i o d o - ~ tyrosine was almost completely dehalogenated, 3-iodo-~tyrosine, 3 - i o d o - a - m e t h y l - ~ ~ - t y r o s i nand e , 3-iOdOtyramine were not, indicating a high degree of structural specificity for these deiodinases. In a subsequent study performed in rats injected with 11 of these compounds, a similar pattern of deiodination specificity was reported (28). The relevance of these results to the catabolism of label from radioiodinated MAbs is unclear, particularly since in the in vitro study it was reported that N-acetyl3,5-diiodo-~-tyrosine,the compound with the closest structural similarity to an iodinated tyrosine residue on a protein (Chart 11), was inert to dehalogenation (25). Experiments are currently underway investigating the catabolism of label from a series of aromatic iodides and their MAb conjugates in order to be able to design better acylation agents for the radioiodination of MAbs. ACKNOWLEDGMENT
The excellent technical assistance of Donna Affleck and Susan Slade is greatly appreciated. Ann Tamariz provided editorial assistance. This research was supported by National Institutes of Health Grants CA 42324, NS 20023, CA 14236, and by Grant DEFG05-89ER60789 from the Department of Energy. LITERATURE CITED (1) Larson, S. M., and Carrasquillo, J. A. (1988) Advantages of radioiodine over radioindium labeled monoclonal antibodies for imaging solid tumors. Nucl. Med. Biol. 15, 231-233. (2) Delaloye, B., Bischof-Delaloye, A., Buchegger, F., von Fliedner, V., Grob, J.-P., Volant, J.-C., Pettavel, J., and Mach, J.P. (1986) Detection of colorectal carcinoma by emissioncomputerized tomography after injection of 123I-labeledFab or F(ab’)2 fragments from monoclonal anti-carcinoembryonic antigen antibodies. J . Clin. Znoest. 77, 301-311. (3) Fracker, P. J., and Speck, J. C. (1987) Protein and cell membrane iodination with a sparingly soluble chlornmide
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