Bioconjugate Chem. 1997, 8, 724−729
724
Method for Radioiodination of Proteins Using N-Succinimidyl 3-Hydroxy-4-iodobenzoate Ganesan Vaidyanathan,* Donna J. Affleck, and Michael R. Zalutsky Department of Radiology, Duke University Medical Center, Durham, North Carolina, 27710. Received April 3, 1997X
A conjugation method has been developed for the radioiodination of proteins which should be adaptable to kit formulation. m-Hydroxybenzoic acid was converted to 3-hydroxy-4-[131I]iodobenzoic acid in 65% radiochemical yield using Chloramine-T as the oxidant. This intermediate was then converted to N-succinimidyl 3-hydroxy-4-[131I]iodobenzoate ([131I]mSHIB) in 75% yield by reaction with Nhydroxysuccinimide and dicyclohexylcarbodiimide in a reaction time of only 10 min. Monoclonal antibody (mAb) 81C6 was labeled in 40-60% yield by reaction with [131I]mSHIB. Performing purifications of radioiodinated compounds using cartridges instead of HPLC did not alter conjugation efficiency, mAb immunoreactivity, or tissue distribution. Thyroid uptake of labeled mAb was low but up to 2.4 times higher than that seen when the mAb was labeled with N-succinimidyl 3-[125I]iodobenzoate. These results suggest that [131I]mSHIB may be a useful reagent for the radioiodination of proteins, particularly in contexts when less complicated purification methods would be advantageous.
INTRODUCTION
For many years, radioiodinated proteins have served as valuable tools for basic biochemical research. An advantage of this approach for labeling proteins is the availability of multiple γ-emitting iodine radionuclides including 125I and 131I, thus permitting direct comparisons, for example, of the tissue distribution of two proteins in the same animal. In the clinical sector, monoclonal antibodies (mAbs1) labeled with 131I, 123I, and 124I have received considerable attention for the diagnosis and treatment of many types of cancer. Because of its simplicity, the most common technique used for the radioiodination of proteins is direct iodination using an oxidant, which results in the introduction of iodine in the meta position of the benzene ring of tyrosine residues (Hunter and Greenwood, 1962; Fraker and Speck, 1978). Although these methods are easy to perform in high radiochemical yield, at least two problems have been associated with their use. First, the strong oxidizing conditions may compromise the biological function of proteins and alter the antigen binding capacity of mAbs (Hayes et al., 1988). Second, the chemical similarity of the protein iodination site to thyroid hormones renders directly labeled proteins susceptible to dehalogenation in vivo due to endogenous deiodinases. To circumvent these problems, conjugation labeling agents have been developed for the radioiodination of proteins; in general, these are reactive with the -amino group of lysine residues [reviewed in Wilbur (1992)]. The prototypical compound is the Bolton-Hunter reagent, N-succinimidyl 3-(4′-hydroxy-3′-iodophenyl)propionic acid * Address correspondence to this author at Box 3808, Department of Radiology, Duke University Medical Center, Durham, NC 27710 [telephone (919) 684-7811; fax (919) 684-7122; e-mail
[email protected]]. X Abstract published in Advance ACS Abstracts, August 15, 1997. 1 Abbreviations: mAb, monoclonal antibody; SIB, N-succinimidyl 3-iodobenzoate; pHBA, p-hydroxybenzoic acid; pHIBA, 3-iodo-4-hydroxybenzoic acid; pSHIB, N-succinimidyl 3-iodo4-hydroxybenzoate; mHBA, m-hydroxybenzoic acid; mHIBA, 4-iodo-3-hydroxybenzoic acid; mSHIB, N-succinimidyl 4-iodo3-hydroxybenzoate; NHS, N-hydroxysuccinimide; DCC, N,N′dicyclohexylcarbodiimide; DCU, N,N′-dicyclohexylurea.
S1043-1802(97)00050-5 CCC: $14.00
(Bolton and Hunter, 1973). Proteins and peptides labeled via the Bolton-Hunter reagent often have exhibited improved biological function as well as a lower degree of deiodination compared with directly radioiodinated proteins (Vaidyanathan and Zalutsky, 1990). On the other hand, conjugation efficiencies for the Bolton-Hunter reagent have been low, generally between 15 and 30% (Wood et al., 1975; Bolton et al., 1976; Vaidyanathan and Zalutsky, 1990). More recent approaches have utilized electrophilic destannylation to label a variety of Nsuccinimidyl esters, which are similar to the BoltonHunter reagent except that they lack a hydroxy group on the benzene ring (Wilbur et al., 1989; Zalutsky and Narula, 1987; Zalutsky et al., 1989). The absence of the hydroxy group ortho to the iodination site is thought to account for the up to 100-fold reduction in thyroid accumulation (an indicator of deiodination) achieved when mAbs are radioiodinated using these methods. Coupling of N-succinimidyl 3-iodobenzoate (SIB) to proteins proceeds in about 2-fold higher yield than the BoltonHunter reagent (Vaidyanathan and Zalutsky, 1990). However, the advantages of SIB and similar reagents have yet to be demonstrated in the clinical domain. Routine application of reagents such as SIB is hindered by the lack of commercial availablity of its N-succinimidyl 3-(tri-n-butylstannyl)benzoate precursor and the need for HPLC purification of SIB to obtain maximum immunoreactivity and coupling efficiency (Garg et al., 1989). HPLC purification of SIB would be particularly problematic for clinical radioimmunotherapy applications, for which preparation of hundreds of millicuries of 131I-labeled mAb frequently is required (Press et al., 1993). It is thus desirable to develop alternative protein radioiodination agents that can be prepared in high yield using simple procedures, while generating a labeled mAb with high immunoreactivity and substantial in vivo stability. With this goal in mind, the current study was undertaken to investigate the potential utility of Nsuccinimidyl 3-hydroxy-4-iodobenzoate (mSHIB) (Chart 1) for the radioiodination of proteins. This reagent could be prepared from commercially available starting materials without HPLC purification, and a mAb could be labeled using mSHIB in reasonable yield and with good in vivo stability. © 1997 American Chemical Society
Radioiodination with mSHIB Chart 1. Structure of Radioiodination Moiety/Site
EXPERIMENTAL PROCEDURES
General. All reagents were purchased from Aldrich unless otherwise specified. Radioiodinated SIB was prepared from N-succinimidyl 3-(tri-n-butylstannyl)benzoate using previously described procedures (Zalutsky and Narula, 1987; Garg et al., 1989). Sodium [125I]iodide and sodium [131I]iodide were obtained from Du Pont-New England Nuclear (North Billerica, MA) in 0.1 N NaOH solution. Murine mAb 81C6, obtained as a gift from Dr. Darell Bigner (Department of Pathology, Duke University Medical Center), is of the IgG2b isotype and reacts with an epitope of the extracellular matrix antigen tenascin (Bourdon et al., 1983). Melting points were determined on Fisher-Johns melting point apparatus and were uncorrected. NMR spectra were obtained with a General Electric Midfield GN-300 spectrometer. Tetramethylsilane (δ ) 0) was used as an internal reference. Mass spectra were obtained on a ZAB-E high-resolution mass spectrometer (VG, Manchester, England), on a Hewlett-Packard GC/MS/DS Model HP-5988A instrument, or on a JEOL SX-102 highresolution mass spectrometer. HPLC was performed with two LKB Model 2150 pumps, an LKB Model 2152 control system, an LKB Model 2138 fixed-wavelength UV detector, and a Beckman Model 170 radioisotope detector. Peak analysis was performed with a Nelson Analytical software package on an IBM computer. Preparation of N-Succinimidyl 3-Hydroxy-4-iodobenzoate. A solution of 9.2 g of potassium iodide and 2.92 g of iodine in 23 mL of water was added over a period of 10 min to a solution of m-hydroxybenzoic acid (3.3 g; 0.024 mol) in 545 mL of 30% ammonium hydroxide. The mixture was stirred at room temperature for about an hour and concentrated to about 80 mL by rotary evaporation. The pH of the solution was adjusted to 4 with concentrated hydrochloric acid, yielding a thick precipitate. This precipitate was extracted with ethyl acetate, and the ethyl acetate solution was washed with brine, dried, and evaporated to yield 1.7 g (27%) of a solid. An analytical sample was obtained by crystallization from a 1:1 mixture of ethanol and water: mp 225-226 °C (decomposes); TLC (86:1:5 chloroform/methanol/acetic acid) Rf ) 0.75 vs 0.63 for starting material; NMR(CD3CN) δ 7.24 (dd, 1H; H-6; JHH ortho ) 8.25 Hz, JHH meta ) 1.65 Hz), 7.43 (d, 1H; H-2; JHH meta ) 1.8 Hz), 7.83 (d, 1H; H-5; JHH ortho ) 8.1 Hz). Verification that this iodinated intermediate was 3-hydroxy-4-iodobenzoic acid was accomplished by heteronuclear multibond connectivity
Bioconjugate Chem., Vol. 8, No. 5, 1997 725
(HMBC) NMR (Bax and Summers, 1986): MS (CI; NH3) 282 [(M + NH4)+], 265 (MH+), 246. Dicyclohexylcarbodiimide (126 mg; 0.6 mmol) was added to a mixture of 3-hydroxy-4-iodobenzoic acid (134 mg; 0.51 mmol) and N-hydroxysuccinimide (67 mg; 0.58 mmol) in 5 mL of dry THF, and the mixture was stirred overnight at room temperature. The precipitated urea derivative was filtered off and washed with THF. The contents of the filtrate were adsorbed onto silica gel (1-2 g), and the product was isolated by silica gel chromatography using ethyl acetate as the eluent (Rf ) 0.61 vs 0.2 for starting material in TLC using this eluent): yield 124 mg (68%); mp 195-197 °C; NMR (CD3CN) δ 2.84 (s, 4H; -CH2CH2-), 7.34 (dd, 1H; H-6; JHH ortho ) 8.10 Hz, JHH meta ) 1.5 Hz), 7.18 (d, 1H; H-2; JHH meta ) 1.8 Hz), 7.96 (d, 1H; H-5; JHH ortho ) 8.10 Hz), 8.25 (br s, 1H; OH); HRMS (FAB+) calcd for C11H9INO5 (MH+) 361.9525, found 361.9542. N-Succinimidyl 3-Hydroxy-4-[125/131I]iodobenzoate Preparation Using HPLC Purification. To simplify purification, minimization of mHBA would be desirable, and so the yield for this reaction was studied as a function of mHBA concentration. Radiolabeling was performed as follows: To 0.1-2.1 mCi of 125/131I in 3 µL or less of 0.1 N NaOH was added 5 µL of m-hydroxybenzoic acid (mHBA) solution (final concentration 0.2 -6.3 mM) in 0.05 N NaOH and 5 µL of 0.04 M solution of Chloramine-T in 0.1 M borate, pH 8.5. The pH of this final mixture was 9.0-9.5. After 10 min of incubation at room temperature, the reaction was quenched by adding 7 µL of 0.4 M sodium bisulfite in 0.1 M borate. The product was purified by reversed-phase HPLC using a Waters µBondapak C18 column (3.9 × 300 mm) eluted with 110: 89:1 (v/v/v) methanol/water/acetic acid at a flow rate of 0.5 mL/min (tR ) 17-19 min vs 8.5 min for mHBA). The HPLC fractions containing the product activity (70% radiochemical yield) were evaporated with an argon purge to remove most of the methanol and acidified with 1 N HCl to a pH of about 1. The radioactive product was extracted into ethyl acetate, and the ethyl acetate solution was washed with brine and dried with sodium sulfate. After being concentrated to a small volume, the solution containing the radioiodinated acid was transferred to a 0.5-dram vial and evaporated to dryness. Esterification of [125/131I]mHIBA was achieved by addition of 30 µL each of 0.1 M solutions of N-hydroxysuccinimide and DCC in dry ethyl acetate (Sure seal, Aldrich). To minimize the duration of the labeling procedure, the yield for the esterification step was investigated as a function of incubation time. The product was purified by silica gel HPLC using an Alltech Partisil silica column (4.6 × 250 mm) eluted with 70:30:2 (v/v/v) hexane/ethyl acetate/acetic acid at a flow rate of 1 mL/min (tR ) 25 min). The optimized radiochemical yield for this step based on HPLC was 85-90%. N-Succinimidyl 3-Hydroxy-4-[125/131I]iodobenzoate Preparation Using Solid-Phase Cartridge Purification. About 2-3 mCi of 125/131I was evaporated to dryness in an Eppendorf tube, and 5 µL of a 2.6 mM solution of mHBA in 0.05 N NaOH was added followed by 5 µL of a 0.04 M solution of Chloramine-T in 0.1 M borate, pH 8.5. After 10 min of incubation at room temperature, the reaction was quenched by adding 7 µL of 0.4 M sodium bisulfite in borate. For the purification of mHIBA, a Waters tC18 ENV reversed-phase solid-phase cartridge activated with methanol and water (6 mL each) was used. The reaction mixture was loaded onto the cartridge and eluted in sequence with 0.5 mL of water, 2 mL of 5:95:1 methanol/water/acetic acid, 3.5 mL of 10:9:1 methanol/ water/acetic acid, 2.5 mL of 50:50:1 methanol/water/acetic
726 Bioconjugate Chem., Vol. 8, No. 5, 1997
acid, and finally, several 0.25 mL portions of methanol. The required [125/131I]mHIBA generally eluted in the third through the sixth methanol fractions. The radiochemical yield was 65-70%, and the HPLC of an aliquot indicated a major single peak corresponding to mHIBA; no mHBA was detected. After these fractions were pooled and the methanol was evaporated, the [125/131I]mHIBA was redissolved in ethyl acetate and dried with sodium sulfate and the esterification reaction was performed as described in the previous section. Purification of the ester was achieved by passing the reaction mixture through a silica Sep-Pak (Waters) activated with 2 mL each of ethyl acetate and hexane and then eluted with 2 mL of hexane, 5 mL of 5% ethyl acetate in hexane, 7 mL of 10% ethyl acetate in hexane, 2 × 2 mL of 50% ethyl acetate in hexane, and, finally, several 1 mL portions of ethyl acetate. The required activity generally eluted in the second 50% ethyl acetate/hexane and the first few ethyl acetate fractions. The radiochemical yield for esterification was 75-80%, and the product was >95% radiochemically pure by HPLC. Radiolabeling of mAb 81C6 with [125/131I]mSHIB. The HPLC or Sep-Pak fractions containing [125/131I]mSHIB were evaporated with an argon stream to a small volume, transferred to a 0.5-dram vial, and evaporated further to dryness. A solution of 81C6 (5 mg/mL; 75 µL) in 0.2 M borate buffer, pH 9.0, was added, and the mixture was incubated for 15-20 min at room temperature. Unreacted [125/131I]mSHIB was quenched by the addition of 0.3 mL of 0.2 M glycine in the above borate buffer, and the incubation was continued for another 5 min. The labeled mAb was purified by gel filtration using a Sephadex G-25 column and phosphate-buffered saline as the eluent. The protein-associated activity was determined by TCA precipitation, and the purity of the labeled protein was further assessed by gel filtration HPLC. For the latter, a Bio-Rad 600 mm × 7.5 mm Bio-Sil SEC-250-10 column was eluted with phosphate-buffered saline, pH 7.14, at a flow rate of 1 mL/min. Immunoreactive Fraction. The immunoreactive fraction of 81C6 labeled with HPLC-purified [131I]mSHIB was compared in a paired-label format to mAb labeled with [125I]mSHIB purified using the cartridge procedure. Approximately 100 ng of each preparation was incubated with serial dilutions of tenascin-positive D-54 MG human glioma homogenates at 4 °C for 18-20 h. The homogenates were washed three times with phosphate-buffered saline containing 2% human serum albumin, and the 125I and 131I activity in the homogenate and supernatant was counted in an automated gamma counter (LKB 1282, Wallac, Finland) using a dual-label protocol. The immunoreactive fraction was determined according to the method of Lindmo et al. (1984). Tissue Distribution Measurements. These studies were performed in a paired-label format in male BALB/c mice weighing about 25 g. In the first study, the biodistribution of 81C6 labeled with HPLC-purified [131I]mSHIB was compared with that labeled with [125I]SIB. Mice were injected intravenously with 8 µCi (5 µg) of [125I]SIB-labeled 81C6 and 6 µCi (5 µg) of [131I]mSHIB-labeled 81C6. Groups of five animals were killed by halothane overdose at 24, 48, 120, 144, and 168 h. In the second study, the tissue distributions of 81C6 labeled with HPLC-purified and Sep-Pak-purified mSHIB were compared. Mice were injected with 7 µCi (10 µg) of 81C6 labeled with HPLC-purified [125I]mSHIB and 5 µCi (10 µg) of Sep-Pak-purified [131I]mSHIB, and tissue uptake was determined at 24, 48, 60, and 168 h postinjection. At necropsy, tissues of interest were isolated, washed, blot-dried, and weighed. Tissues were then counted for
Vaidyanathan et al. 125 I and 131I in a gamma counter using a dual-label counting program. The percent injected dose per gram of tissue was calculated by comparison to standards of appropriate count rate. Since the tissue distribution was performed in a paired-label format, with each animal serving as its own control, a paired t test was used to determine the statistical significance of differences between the uptake of the two radionuclides.
RESULTS AND DISCUSSION
Direct radioiodination is an efficient, convenient approach for protein labeling and, as a result, is the most commonly used technique for basic and clinical applications. This remains the case despite its obvious drawbacks. Exposure of the protein to oxidants can compromise biological function and, for some mAbs, lower immunoreactivity (Hayes et al., 1988). For in vivo applications, dehalogenation can result in loss of label from directly labeled proteins, decreasing their ability to deliver radiation to specific cell populations. An alternative is to use the Bolton-Hunter reagent, which is commercially available labeled with 125I and thus is only suitable for in vitro studies. The presence of the two methylene groups between the ester functionality and the iodine-substituted benzene ring decreases the hydrolytic stability of this reagent, resulting in only modest conjugation yields and, possibly, in vivo susceptibility to hydrolytic enzymes (Vaidyanathan and Zalutsky, 1990). While reagents such as SIB have shown encouraging results for the radioiodination of proteins (Zalutsky et al., 1989; Wilbur et al., 1989), their chemistry may not be readily adaptable for widespread use, particularly in centers without access to radio-HPLC. Reagents such as SIB are synthesized via destannylation to avoid the need for a hydroxy group ortho to the iodination site to activate the ring toward electrophilic substitution. While this hydroxy group decreases the strength of the carbon-iodine bond, it does not always lead to rapid deiodination in vivo. For example, we have reported that a mAb labeled using the BoltonHunter reagent exhibited considerably lower thyroid accumulation than the same mAb labeled using Iodogen (Vaidyanathan and Zalutsky, 1990). This motivated us to investigate pSHIB, which is analogous to the BoltonHunter reagent except it does not contain the two intervening methylene groups (Vaidyanathan et al., 1993). The deiodination of a mAb labeled using pSHIB was only slightly inferior to that of the same mAb labeled with SIB. Unfortunately, the yields for coupling pSHIB to mAbs were modest even with long reaction times. It was hypothesized that these poor coupling efficiencies were due to the presence of a hydroxy group at the para position of the carboxylic ester group. The current study was performed to investigate this possibilty through the synthesis of the isomeric compound mSHIB in which the hydroxy group was moved to the meta position. In addition, the adaptability of m[131I]SHIB for preparation by a kit method was studied. HPLC standards of mHIBA and mSHIB were prepared following a procedure reported for the isomeric compound (Vaidyanathan et al., 1993). Unlike the case for pHBA, iodination of mHBA can result in three different monoiodinated regioisomers. Proof that the iodinated product indeed was 3-hydroxy-4-iodobenzoic acid was provided by a HMBC NMR experiment (Bax and Summers, 1986). Since the ultimate goal of this work is to develop a method that is adaptable to kit formulation, it is essential to minimize impurities that may have deleterious effects on the peptide or mAb. For example, the active ester of m-hydroxybenzoic acid itself could react with lysine
Radioiodination with mSHIB
Bioconjugate Chem., Vol. 8, No. 5, 1997 727
yields of 60-65%. In comparison, 3.9 mM pHBA had been used previously for the preparation of 4-hydroxy3-[131I]iodobenzoic acid (Vaidyanathan et al., 1993). Conversion of 4-hydroxy-3-[131I]iodobenzoic acid to the corresponding NHS ester required 2-3 h (Vaidyanathan et al., 1993). The esterification of 3-hydroxy-4-[131I]iodobenzoic acid was studied as a function of time. Radiochemical yields of >75% were obtained in as little as 5-10 min (Figure 1B). No further improvement in yield was achieved by increasing the reaction time up to 1 h. Although different solvents were used in the two studies (ethyl acetate, mHIBA; THF, pHIBA), the faster kinetics observed for the meta isomer may be a result of relatively higher susceptiblity of carboxyl carbonyl in 3-hydroxy-4-iodobenzoic acid toward nucleophilic attack (vide infra). One of the principal advantages of mHIBA compared with pHIBA is the possible higher reactivity of the active ester of the former with lysine -amino groups on proteins. As mentioned before, poor coupling yields were obtained for the conjugation of pSHIB to mAb 81C6, and this was attributed to the influence of the hydroxy group at the para position (Vaidyanathan et al., 1993). On this basis, it was predicted that higher yields would be obtained with mSHIB, and, in fact, conjugation efficiencies of 40-60% were observed when mAb 81C6 was incubated with [131I]mSHIB for 20 min. Increasing the incubation time up to 1 h did not result in any significant increase in conjugation efficiency. In comparison, only 10-15% coupling yield was obtained at 1 h with pSHIB, confirming the prediction that mSHIB would offer better conjugation yields. The characteristics of 81C6 labeled with mSHIB prepared and purified using HPLC were compared to those obtained using the cartridge procedure. Size exclusion HPLC demonstrated the presence of a single radioactive peak with an elution time corresponding to that of IgG. There was no shoulder on the peak, indicating the absence of higher molecular weight aggregates in the preparations. Previous studies with SIB have shown that it is important to minimize cold active ester impurities to maintain mAb immunoreactivity (Zalutsky and Narula, 1988). The immunoreactive fractions for the binding of 81C6 labeled to D-54 MG human glioma homogenates were excellent. When the mAb was reacted with mSHIB prepared using HPLC, an immunoreactive fraction of 95% was obtained compared with 91% with the cartridge procedure. This is consistent with mass sprectroscopy and IR analyses which did not detect any NHS ester of mHBA in the mSHIB preparation (data not shown). The tissue distribution of 81C6 mAb labeled with HPLC-purified [131I]mSHIB was compared in normal
Figure 1. (A) Radiochemical yield for the preparation of [131I]mHIBA as a function of mHBA concentration. (B) Temporal effect on the coupling efficiencies of [131I]mSHIB to 81C6.
-amino groups and, if present in considerable amounts, could alter its biological characteristics. To determine the minimum amount of mHBA needed to have a satisfactory radiochemical yield, the radioiodination of mHBA was studied as a function of mHBA concentration. The radiochemical yield for HIBA increased with mHBA concentration, but the concentration dependence was not dramatic (Figure 1A). While a yield of 43.5 ( 10.5% was obtained with 0.2 mM mHBA, a yield of 62.3 ( 3.5% was obtained when the concentration was 6.3 mM (p < 0.05). An mHBA concentration of 1.3 mM was chosen for subsequent radioiodinations, and this gave radiochemical
Table 1. Paired-Label Tissue Distribution of Radioiodine in Normal Mice following Injection of 81C6 Labeled with [125I]SIB and with [131I]mSHIB % injected dose per organa 1 day
5 days
6 days
tissue
SIB
mSHIB
SIB
mSHIB
SIB
mSHIB
liver spleen lungs heart kidneys stomach sm intest lg intest muscle bone blood brain
4.29 ( 0.51 0.34 ( 0.04 1.97 ( 0.46 0.53 ( 0.10 1.83 ( 0.18 0.50 ( 0.07 2.39 ( 0.27 1.06 ( 0.13 10.55 ( 1.67 5.55 ( 0.91 20.93 ( 1.62 0.17 ( 0.03
4.10 ( 0.50 0.29 ( 0.03 2.26 ( 0.62b 0.57 ( 0.11 1.77 ( 0.19b 0.48 ( 0.06b 2.04 ( 0.24 1.00 ( 0.06b 10.72 ( 1.73b 4.64 ( 0.76 26.82 ( 1.93 0.20 ( 0.03
1.59 ( 0.18 0.12 ( 0.02 0.85 ( 0.38 0.19 ( 0.03 0.57 ( 0.07 0.13 ( 0.01 0.78 ( 0.13 0.33 ( 0.04 4.06 ( 0.64 .88 ( 0.27 7.12 ( 0.84 0.06 ( 0.01
3.24 ( 0.35 0.19 ( 0.03 2.08 ( 0.97 0.43 ( 0.05 1.21 ( 0.15 0.26 ( 0.04 1.42 ( 0.22 0.65 ( 0.10 8.56 ( 1.09 3.13 ( 1.57 18.99 ( 1.57 0.13 ( 0.02
1.23 ( 0.11 0.08 ( 0.01 0.41 ( 0.06 0.12 ( 0.03 0.44 ( 0.04 0.09 ( 0.01 0.59 ( 0.08 0.26 ( 0.04 2.74 ( 0.26 1.26 ( 0.07 5.11 ( 0.48 0.04 ( 0.00
3.08 ( 0.44 0.17 ( 0.02 1.16 ( 0.19 0.35 ( 0.10 1.15 ( 0.17 0.23 ( 0.05 1.33 ( 0.22 0.62 ( 0.12 7.23 ( 0.43 2.59 ( 0.20 16.82 ( 1.74 0.11 ( 0.02
a
Mean ( SD (n ) 5). b The difference between two values statistically not significant by a t test (p > 0.05).
728 Bioconjugate Chem., Vol. 8, No. 5, 1997
Vaidyanathan et al.
Figure 2. Paired-label thyroid uptake of 131I and 125I following injection of 81C6 labeled with [131I]mSHIB and [125I]SIB in normal mice.
mice with that for mAb labeled with [125I]SIB. SIB was chosen for comparison because use of this reagent reduced dehalogenation and normal tissue levels and increased tumor uptake and therapeutic efficacy compared with mAb labeled via the Iodogen method (Zalutsky et al., 1989; Schuster et al., 1991). The paired-label thyroid uptake of 81C6 labeled with [131I]mSHIB and with [125I]SIB is shown in Figure 2. Initially, the difference in uptake of 131I and 125I was small but statistically significant (p < 0.05). With time the difference increased and by day 7, the percent injected dose (ID) of 131I was 2.5-fold higher than that of 125I. Nonetheless, thyroid accumulation of radioiodine from mAb labeled using mSHIB was
