Bioconjugate Chem. 1990, 1, 387-393
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Radioiodination of Antibodies via N-Succinimidyl 2,4-Dimethoxy-3-(trialkylstannyl)benzoates Ganesan Vaidyanathan and Michael R. Zalutsky’ Duke University Medical Center, Department of Radiology, Box 3808, Durham, North Carolina 27710. Received August 20, 1990
We have previously shown that use of N-succinimidyl3-iodobenzoate(SIB) for radioiodination of monoclonal antibodies (MAbs) decreases the loss of radioiodine in vivo compared to MAbs labeled by using conventional methods. Herein, the synthesis of N-succinimidyl 2,4-dimethoxy-3-(trialkylstannyl)benzoates (alkyl = Me, Bu) are described as is their use as precursors for the radiosynthesis of N-succinimidyl2,4-dimethoxy-3-iodobenzoate (SDMIB). A MAb F(ab’)2 fragment labeled with SDMIB retained its ability to bind specifically to tumor homogenates. Paired-label tissue distribution studies indicate that the thyroid uptake (an indicator of deiodination) of hydrolyzed SDMIB was about 20 times that of hydrolyzed SIB. In contrast, thyroid uptake for SDMIB, when conjugated to a MAb, was only 1.42.8 times that for SIB and was considerably lower than levels reported in the literature for MAbs labeled by using direct, electrophilic iodination methods. Although MAbs labeled with SDMIB are significantly more inert to dehalogenation than those labeled by conventional methods, compared to the original SIB reagent, addition of two methoxy groups decreased retention of label in vivo.
When radiolabeled monoclonal antibodies (MAbs)’ are administered t o patients intravenously, uptake of radioactivity is low, generally about 0.005%of the injected dose per gram (I). For this reason, single photon emission tomography is often required t o obtain satisfactory sensitivity for tumor detection (2). Of the nuclides under active investigation for use in radioimmunoscintigraphy, 6-h 99”Tcand 13-h 1231have the most suitable nuclear properties for single photon emission tomography, particularly when quantitation of tracer uptake for dosimetric calculations is required. For many applications of labeled MAbs, the added expense and inconvenience of using 1231 may be more than offset by the greater compatability of its physical half-life with MAb pharmacokinetics. Methods for the direct radioiodination of proteins have been available for many years (3). However, use of oxidants such as Iodogen or chloramine-?‘ results primarily in the formation of iodinated tyrosine residues ( 4 ) ,and MAbs labeled by this approach have been observed to undergo extensive loss of label in vivo (5-7). Since numerous deiodinases exist with varying specificities for iodotyrosines and thyronines @-IO), MAb dehalogenation probably is related to recognition of iodotyrosines on the MAb by these enzymes. In an attempt to minimize MAb dehalogenation by providing an iodination site with a different chemical structure, we developed a method which involves reaction of the MAb with N-succinimidyl3-iodobenzoate(SIB, 1, Chart I), which is prepared by the iododestannylation of N-succinimidyl 3-(tri-n-butylstannyl)benzoate(ATE, 2) Abbreviations: MAbs, monoclonal antibodies; SIB, N-succinimidyl 3-iodobenzoate; ATE, N-succinimidyl 3-(tri-nbutylstanny1)benzoate; DMATE-Bu, N-succinimidyl 2,4dimethoxy-3-(tri-n-butylstannyl)benzoate;DMATE-Me, N-succinimidyl2,4-dimethoxy-3-(trimethylstannyl)benzoate;DCC, dicyclohexylcarbodiimide; SDMIB, N-succinimidyl2,4-dimethoxy34odobenzoate; IBA, 3-iodobenzoicacid; DMIBA, 2,4-dimethoxy3-iodobenzoic acid.
Chart I COOR
t
NHS
1.
SIB R E M , X = Z = H , Y = I
2.
ATE R=NHS, X = Z = H , Y = S n B u 3
3a. DMATE-Me R = NHS, X = Z = OCH,, Y = SnMq
3b. DMATE-Bu R = NHS, X = Z = O m 3 ,Y = SnBu, 4.
SDMIB R+EIS,
X=Z=OCH,, Y = I
5. IBA R = X = Z = H , Y - I 6. DMIBA R = H , X=Z=OCH3, Y = I (11). Paired-label studies demonstrated that proteins labeled via the ATE method had significantly lower uptake of activity in the thyroid and stomach (tissues known to avidly accumulate free iodide) compared to the same proteins labeled by using the Iodogen method (11-13). Similar results have been reported by other groups using MAbs and fragments labeled with 4-iodophenyl conjugates (14,15),confirming the influence of the nature of the MAb iodination site on in vivo stability. Further improvements in protein radiohalogenation methodology will be facilitated by gaining a better understanding of the structural features required for minimizing dehalogenation. For example, although both the Bolton-Hunter and Iodogen methods involve radioiodination ortho to a hydroxyl group on an aromatic ring,
1043-1802/90/2901-0387$02.50/0 0 1990 American Chemical Society
388
Bioconjugate Chem., Vol. 1, No. 6, 1990
MAbs labeled by using the Bolton-Hunter method are considerably more inert to dehalogenation in vivo (16). In the present study, we investigated the utility of labeling MAbs via the iododestannylation of N-succinimidyl 2,4dimethoxy-3-(tri-n-butylstannyl)benzoate(DMATEBu, 3b) (17) and its trimethylstannyl analogue 3a (DMATE-Me). The primary objective of this investigation was to determine whether substitution of electron-rich methoxy groups ortho to the iodine on the aromatic ring would decrease the probability of nucleophilic displacement of the iodine, thereby enhancing the in vivo stability of the carbon-iodine bond. EXPERIMENTAL PROCEDURES
General Procedures. NMR spectra were obtained in CDC13 solution with a General Electric midfield GN-300 spectrometer. The proton chemical shifts are reported in ppm downfield from internal TMS (0.00 ppm). IR spectra were obtained on a BOMEM MB-100 variable-resolution FTIR spectrophotometer. Mass spectral data were obtained on a VG 70s (VG Analytical, Danvers, MA) instrument operating in the E1 mode. Melting points were determined on a Haake Buchler variable-heat apparatus and are uncorrected. All reagents were of reagent grade or better. THF was distilled over LiAlH4. ATE was prepared as reported before (11). DMATE-Bu was synthesized with slight modifications of the previously described procedure ( I 7), and DMATE-Me was prepared in a similar fashion, the details of which are given below. Sodium [1251]iodideand sodium ['3lI]iodide, both in 0.1 N NaOH, were obtained from Du Pont-New England Nuclear (North Billerica, MA). MAb CllO is a murine IgGl reactive with carcinoembryonic antigen (18)and was obtained as a gift from Dr. David Johnson of Abbott Laboratories. Mel-14 is a murine IgG2a reactive with the tumor-associated chondroitin sulfate present in melanomas and gliomas (19). The procedure for generation of Mel1 4 F(ab')z fragments has been described (20). This fragment was obtained as a gift from Dr. Dare11 D. Bigner of the Department of Pathology, Duke University Medical Center. Thin-layer chromatography was done on EM Science analytical silica plates. Flash chromatography was done with 230-400 mesh silica gel from VWR Scientific, Marietta, GA. High-pressure liquid chromatography was conducted with an LKB Model 2150 pump, an LKB Model 2151 variable-wavelength UV detector, and a Beckman Model 170 radioisotope detector. Peak analysis was performed with a Nelson analytical software package on an AT&T 6300 computer. The column used was an Alltech silica gel column (Partisil 10 silica 10 pm, 250 X 4.6 mm). Radioactivity counting was performed with a LKB 1282 dual channel y-counter. Synthesis of DMATE-Me (3a). A solution of %bromo2,4-dimethoxybenzoic acid, prepared as previously described (17) (786 mg, 3 mmol) in 96 mL of dry THF, was cooled in an ether/liquid nitrogen bath (-100 "C). To this solution was added dropwise 3.8 mL of butyllithium (1.6 M in hexane; 2 equiv), and the mixture was stirred at -100 "C for another 30 min. A solution of trimethylstannyl chloride (1.56 g, 7.85 mmol) in THF (5 mL) was added. The reaction mixture was allowed to warm to room temperature and stirred overnight. All of the above operations were done under an argon atmosphere. A t the end of the reaction, the reaction mixture was partitioned between water and ether (100 mL of each). The aqueous layer was further extracted with ether (3 X 50 mL). The
Vaidyanathan and Zalutsky
combined ethereal layer was washed with brine (2 X 10 mL), dried over sodium sulfate, and evaporated to yield 1.2 g of a white solid. This was purified by silica gel flash chromatography with a gradient elution of 0-30% ethyl acetate in hexane to give 460 mg (45 % yield) of a white, crystalline solid. Mp: 108-111 "C. N M R (CDC13) 6 0.37 (s,9 H,SnCH3),3.82 (s,3 H,4-OCH3),3.85 (s,3 H,2-OCH3) 6.75 and 8.14 (dd, 2 H, aromatic, J = 8.65 Hz). (Note: NMR indicates this is the free acid in contrast to the tin ester obtained in the case of the butyl analogue. No attempt was made to further characterize it prior to proceeding to the next step.) To 250 mg (0.72 mmol) of the above solid in 10 mL of dry T H F were added 120 mg (1.04 mmol) N-hydroxysuccinimide and 180 mg (0.87 mmol) of DCC under an argon atmosphere. The mixture was stirred overnight a t room temperature. Precipitated dicyclohexylurea was filtered and washed with THF. The filtrate containing the product was evaporated in a rotary evaporator, and the residue was flash chromatographed on silica gel using a 0-30% ethyl acetate in hexane gradient to give 180 mg (58% yield) of a white, crystalline solid. Mp: 85-86 "C. NMR (CDC13) 6 0.33 (s, 9 H, SnMea), 2.89 (br s, 4 H, NHS), 3.78 (s,3 H), 3.80 (s, 3 H), 6.65 and 8.10 (dd, 1 H each, J = 8.71 Hz). IR: (KBr) cm-l 3328, 2931, 2853,1773,1737,1578. MS: m/z 428 (M - CH3), 329 (M - ONHS), 299. Anal. Calcd for C15HleO,jNSn (M - CH3) 428.0156, found 428.0163. TLC: 30% EtOAc/hexane, Rf = 0.16. HPLC: more than 99% pure; retention volume is 11.2 mL (EtOAc/hexane/HOAc 50:50:2; 0.8 mL/min, 10 bar). Radioiodination Procedure. [1251]SIBwas synthesized from ATE and purified as described before (21). For the synthesis of [131]SDMIB(4), sodium [1311]iodide (ca. 100 pCi) in about 5 pL of 0.1 N NaOH was transferred to a l-mL conical glass vial and evaporated with a gentle stream of argon. (No loss of 1311 activity was observed.) A 3% solution of acetic acid in CHC13, in a volume twice that of the 1311solution, was added, followed by 15 pL of tertbutyl hydroperoxide solution (10% dry basis in CHCl3) and 5 pL (0.5 pmol) of either DMATE-Bu or DMATEMe. The mixture was stirred for 1-30 min. The percent yield of [1311]SDMIBwas determined by injecting an aliquot onto the HPLC and determining the fraction of the 1311which eluted with a retention time corresponding to that of a cold SDMIB standard ( t =~ 14.0 min). For biological experiments, higher amounts (0.5-1.5 mCi) of radioactivity were used. Free acids IBA (5) and DMIBA (6) were prepared by hydrolyzing the corresponding N-succinimidyl esters overnight in phosphate-buffered saline. The extent of hydrolysis was checked by acidifying an aliquot with acetic acid, extracting with EtOAc, and analyzing the EtOAc layer by HPLC. Labeling of MAbs by Reaction with [1251]SIBo r [1311]SDMIB. The general methodology used for MAb labeling was similar to that reported previously (16,21). In brief, the HPLC fractions containing [131I]SDMIBor [1251]SIB(300-1000 pCi) were concentrated to
