Bioconjugate Chem. 1991, 2, 44-49
44
Fluorine-18 Labeling of Monoclonal Antibodies and Fragments with Preservation of Immunoreactivity Pradeep K. Garg, Sudha Garg, and Michael R. Zalutsky* Duke University Medical Center, Department of Radiology, Box 3808, Durham, North Carolina 27710. Received November 5, 1990
A new method is reported for labeling proteins with the positron-emitting nuclide lSF. Initially, 4-[18F]fluorobenzylamine was prepared in two steps from aqueous [lSFlfluoride in high yield. The 'SF acylation agent was formed by reaction of this product with disuccinimidyl suberate. Overall yields for the 4- [1SF]fluorobenzylaminesuccinimidyl ester ([ laF]SFBS), decay corrected to the end of cyclotron bombardment, were about 30 5% in a synthesis time of 60 min. After a 15-min reaction, 30-45 % (decay corrected) of the ['SFISFBS could be coupled to intact antibodies and their F(ab')z and Fab fragments. Coupling yields were dependent on protein concentration but not reaction time. HPLC purification of ['SFISFBS was necessary to obtain optimal coupling efficiency and immunoreactivity. The immunoreactivities of 1sF-labeled F(ab')2 and Fab fragments of an antimyosin antibody were 89 f 5% and 75 f 9 9; , respectively. Biodistribution studies in normal mice demonstrated similar in vivo behavior of 'SF-labeled antibody fragments and those labeled with 1251 by using N-succinimidyl 3-[1251]iodobenzoate. These results indicate that this method may be useful for labeling monoclonal antibodies and other proteins and peptides with lSF.
Positron-emission tomography (PET)' is a functional imaging modality which permits quantitative evaluation of physiology in the living human. Considering the ubiquitousness of proteins and peptides and their role in many disease processes, the development of a method for labeling proteins with a positron-emitting nuclide could provide valuable diagnostic tools. For example, serial PET scans of a monoclonal antibody (MAb) labeled with a positron emitter could be used to quantitate their threedimensional distribution in vivo. In addition to its diagnostic utility, this information could be used to generate accurate dosimetry data from which a radiolabeled MAb therapy procedure could be planned more rationally. Methods for labeling proteins with positron-emitting nuclides have been described in only a few published reports. Proteins have been labeled with 20-min half-life llC by llC methylation (1, 2), and methods for labeling proteins with 68-min 68Ga have also been reported (3-5). Of the commonly available positron emitters, 110-min 18F appears to be the nuclide of choice for labeling MAbs and their fragments. The longer half-life of l8F would facilitate imaging a t later time points, a feature which may be important because of the relatively slow clearance of MAbs from nontarget tissues (6). In addition, Kilbourn and coworkers (7) have reported that because of the lower positron energy of 18F compared to 68Ga,the radiation dose received by critical normal organs would be as much as 40% lower per mCi of administered activity with lSF. A number of approaches for labeling proteins with 18F have been described. Muller-Platz et al. (8) utilized a water-soluble carbodiimide to label urokinase with 18Fvia a 2- [lsF]fluoroacetate intermediate. In another prelim~
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Abbreviations used: PET, positron-emissiontomography; MAb, monoclonal antibody; ['BFISFBS, N-succinimidyl8 4 (4'[lsF]fluorobenzyl)amino]suberate; DSS, disuccinimidyl suberate;PBS, phosphate-buffered saline;SFBS,N-succinimidyl8-[(4'fluorobenzyl)amino]suberate; ID, injected dose. 1043-1802f9 1f2902-0044$02.50/0
inary report, the synthesis of the potential protein labeling and m-maleimagents N-(p-[lsF]fluorophenyl)maleimide ido-N-(p-[lsF]fluorobenzyl)benzamide have been described (9). Two approaches for labeling proteins with '8F have been reported by Kilbourn et al. (7). In the first, methyl 3-['8F]fluor0-5-nitrobenzimidate was synthesized by nucleophilic substitution of [lSF]fluoride for nitro in 3,5-dinitrobenzonitrile, followed by reaction with sodium methoxide in anhydrous methanol. In the second, 4-[lSF]fluorophenacyl bromide was synthesized from [lsF]fluoride ion in three steps beginning with fluoro for nitro exchange on 4-nitrobenzonitrile. Several proteins could be labeled by incubation with either reagent at nearphysiological pH. Of critical importance is the demonstration that proteins can be labeled with lSF without compromising their biological integrity; however, little data is available concerning the effect of 18Flabeling on protein function. The blood clearance of lSF-labeled human serum albumin has been shown to be similar to that observed when lZ5I was used as the label (7),but as these authors point out, human serum albumin cannot be denatured easily. This paper describes a new method for labeling MAb and other proteins with lSFthat utilizes N-succinimidyl 8-[(4'-[1SF]fluorobenzyl)amino]suberate([18F]SFBS) as the lSF-labeled acylation agent. MAb F(ab')z and Fab fragments maintained immunoreactivity after labeling with lSF. The results of biodistribution studies in normal mice suggest that these MAb fragments retain their lSFlabel after administration in vivo. EXPERIMENTAL PROCEDURES General Procedures. 'H NMR spectra were obtained in CDC4 and recorded on a General Electric midfield GN300 spectrometer. Proton chemical shifts are reported in ppm downfield from internal TMS (0.00 ppm). Mass spectral data were obtained on a Finigan 4500 single quadrupole mass spectrometer. Melting points were deter@ 1991 American
Chemical Society
Fluorine-18 Labeling of Antibodies
mined on a Haake Buchler variable-heat apparatus and are uncorrected. Lithium aluminum hydride, 4-nitrobenzonitrile, tertbutylammonium hydroxide, 4-fluorobenzylamine, and dimethyl sulfoxide were obtained from Aldrich (Milwaukee, WI). Disuccinimidyl suberate (DSS) was purchased from Pierce (Rockford, IL). Sodium [1251]iodide was obtained from Du Pont-New England Nuclear (North Billerica, MA). The generation and purification of the murine MAb RllDlO directed against cardiac myosin have been described (10). The Fab and F(ab')2 fragments of this antmyosin MAb were provided as gifts from Dr. David Shealy from Centocor (Malvern, PA). Mel-14 F(ab')2 (11) is reactive with the tumor-associated chondroitin sulfate antigen present in melanomas and has no known reactivity with cardiac myosin. MAb 81C6 is a murine IgG reactive with the extracellular matrix antigen tenascin (12). Both Mel-14 F(ab')z and 81C6 IgG were obtained as gifts from Dr. Dare11 D. Bigner of the Department of Pathology, Duke University Medical Center. Radioactivity levels were assessed with an LKB 1282 dual-channel y-counter. High-pressure liquid chromatography was performed in isocratic mode with an LKB Model 2150 pump, 2151 variable-wavelength UV detector, and a Beckman Model 170 radioisotope detector. A Nelson Analytical software package was used for peak analysis. The separation system used for analysis of aryl fluorides was a Partisil 10 silica 10 pm, 250 X 4.6 mm column (Alltech) eluted with ethyl acetate (or ethyl acetate-hexane) at a flow rate of 0.8 mL/min. For size-exclusion HPLC, a 300 X 7.5 mm Biosil TSK 250 column (Bio-Rad) eluted with phosphate-buffered saline (PBS, pH 7.4) at a flow rate of 1 mL/min was used. Preparation of N-Succinimidyl 8-[ (4'-Fluorobenzy1)aminolsuberate (SFBS). DSS (368 mg) was dissolved in 400 pL of dimethyl formamide. To the resultant clear solution was added 115 mg of 4-fluorobenzylamine and the reaction mixture was stirred at room temperature for 2 h. After the addition of 10 mL of diethyl ether, the precipitate was removed. The filtrate was washed with 1 mL of 0.5 N HC1 followed by 2 mL of water and then dried over Na2S04. After removing the ether by rotary evaporation, the product (205 mg, 55 % yield) was isolated by flash chromatography on a silica gel column using 10% hexane in ethyl acetate as the eluant (melting point 110 "C). Purity was greater than 95 % by TLC [silica gel; ethyl acetate-hexane (9:l); Rf0.61. The assigned structure for SFBS was in agreement with its NMR and mass spectral data. 1H NMR: (CDC13, 6) 1.4 (m, 4 H, alkyl), 1.72 (m, 4 H, alkyl), 2.25 (t, 2 H, CH2COO), 2.6 (t, 2 H, NHCOCH2CH2), 2.78 (s,4 H, N-succ), 4.15 (d, 2 H, ArCH2NH), 7.007.24 (m, 4 H, aryl). MS: m / e 378 (M+,6), 264 (M+- 114, 44), 235 (M+ - 143, 27). Anal. Calcd for C19H23N205Fl/zH20: C, 58.91; H, 6.2; N, 7.20. Found: C, 59.13; H, 5.4; N, 7.05. Preparation of 4-[ 18F]Fluorobenzonitrile. Aqueous [l*F]fluoridewas produced with the 180(p,n)18Freaction by irradiating [180]H20 in a small-volume silver target (13). The synthesis of this compound was achieved by using a modification of the method of Kilbourn et al. (7). The conversion of aqueous [18F]fluoride ion to tetrabutylammonium [18F]fluoride and the fluoro for nitro exchange were performed in the same vessel in order to reduce vessel-to-vessel transfer losses. Briefly, 300 pL of [180]H20containing 5-100 mCi of [lsF]fluoride was added by a remote handling system (14) to a glass tube containing 1 mL of acetonitrile and 2-10 pmol of tetrabutylammo-
Bloconjugate Chem., Vol. 2, No. 1, 1991 45
nium hydroxide. The mixture was evaporated by heating at 110 "C and using a gentle stream of nitrogen. Any remaining water was removed azeotropically with 2 X 1 mL of acetonitrile. To the dried tube was added 800 pg of 4-nitrobenzonitrilein 100 pL of DMSO, and the solution was heatedat 140-150"Cfor 15min. There~ultant4-[~8F]fluorobenzonitrile was separated from the reaction mixture with a Cl8 Sep-Pak (Millipore) eluted with 0.5 mL of diethyl ether. Decay-corrected yields were 6 0 4 0 % . A radiochemical purity of 95-99 ?& was determined by HPLC [silica column; ethyl acetate-hexane (1:9); t~ = 8.8 min]. The crude product was used for the next step. HPLC analyses revealed the presence of less than 50 pg of 4-nitrobenzonitrile ( t R = 12.7 min) in the 4 - [ 1 8 F ] f l ~ ~ r o benzonitrile preparation. Synthesis of 4-[18F]Fluorobenzylamine. Lithium aluminum hydride (50mg) was added to the above product and the mixture was allowed to stand at room temperature for 5 min. The reaction was quenched with 2 mL of aqueous saturated ammonium chloride followed by 2 drops of 1N NaOH. The ether layer was separated and washed with 1 mL of H2O and dried over NazSOr. The decaycorrected yield for this step was 80-95% and the radiochemical purity was determined to be 95-99% by TLC (silica; ethyl acetate). Preparation of [18F]SFBS. DSS is a homobifunctional agent that reacts with primary amino groups (15, 16). To 150 pg of DSS in 15 pL of DMF was added 5-50 mCi of 4-[18F]fluorobenzylamine.The mixture was incubated for 5 min at room temperature, then the ether was evaporated slowly under a stream of nitrogen. In preliminary studies, the residue was used in unpurified form for protein labeling. Subsequently, [18F]SFBSwas purified by HPLC using a silica gel column eluted with ethyl acetate at 0.8 mL/min ( t =~12.5 min). General Method for Labeling Proteins with [lSF]SFBS. The organic solvent (ether, nonpurified; ethyl acetate, HPLC purified) containing the [18F]SFBSwas evaporated, and 200-1100 pg of the protein of interest in 100-200 pL of pH 8.5 borate buffer was added. The mixture was incubated for 15 min at room temperature and the reaction terminated by addition of 300 pL of 0.2 M glycine in 0.1 M borate. The [18F]SFBS-protein conjugate was isolated from lower molecular weight impurities with a 1 X 10 cm Sephadex G-25 column eluted with PBS. Protein-associated radioactivity, determined by precipitation with 20 % trichloroacetic acid, was greater than 96 % for all preparations. Selected batches of labeled proteins were analyzed by size-exclusion HPLC. E f f e c t of I n c u b a t i o n T i m e a n d P r o t e i n Concentration. Because of the short half-life of 18F,the time dependence of the [18F]SFBScoupling reaction was of interest. Antimyosin Fab fragment (500 pg in 200 pL) was added to about 1mCi of [18F]SFBSand incubated at room temperature for 15,30,60,90,or 120 min. To study the effect of protein concentration on coupling efficiency, 100-150 pL of MAb fragment at 2.5,5.5, and 8.5 mg/mL was reacted with about 1 mCi of [18F]SFBS for 30 min. HPLC-purified [18F]SFBSwas used. Protein coupling efficiency was determined by dividing the l8F activity eluting in the void volume of the Sephadex G-25 column by the total 18Factivity added to the column. Fluorine-18 activity levels were measured with a Capintec CRC-7 dose calibrator. Two or three determinations were performed at each set of reaction conditions. Immunoreactivity Assay. The immunoreactivity of '8F-labeled antimyosin Fab and F(ab')2 fragments was determined with a 1-mLmyosin Sepharose column, which
46
Bioconjugate Chert?.,Vol. 2, No. 1, 1991
was obtained as a gift from Dr. David Shealy of Centocor. Approximately 10 ng of 'SF-labeled antimyosin fragment was added to the column; the column was then washed with PBS containing 0.5% human serum albumin and stripped with 8 mL of 0.1 M glycine (pH 2.5). Aliquots (100-250 pL) in triplicate from both the PBS wash and the glycine stripping solution were counted for 18Factivity in an automated y-counter. Percent immunoreactivity was calculated from the ratio of activity in glycine to activity in glycine plus PBS. In some cases, nonspecific binding to the myosin Sepharose column was measured using 18F-labeled Mel-14 F(ab')z. Biodistribution Measurements. Initially, the effect of HPLC purification of the [18F]SFBScoupling agent on the tissue distribution of 18F-labeledantimyosin Fab was studied. With both HPLC-purified and unpurified [lSF]SFBS, 'SF-labeled antimyosin Fab was prepared as described above. Normal BALB/c mice weighing 20-25 g were injected with 1-2 pg (8 pCi) of each preparation and groups of five animals were sacrificed by ether overdose at 0.5,1, and 2 h. Following dissection, tissues of interest were removed, weighed, washed with PBS, and counted for '8F activity. In the second experiment, antimyosin F(ab')z was labeled with by using N-succinimidyl 3-[1251]iodobenzoateas described (17) and with 18Fby using HPLC-purified [l8F]SFBS. Mice were injected with 3 pg (3 pCi) of Iz5I-labeledFab and 2 pg (4 pCi) of 18F-labeled Fab, and the tissue distribution of both nuclides was determined at 0.5,1, and 2 h. Counting data for lz51were corrected for crossover of 18Finto the lz51counting window. The percent injected dose (ID) in each tissue 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 the tissue distribution of Fab labeled by reaction with HPLC-purified and unpurified ['8F]SFBS were made by using the Student's t test (18). Since the experiment comparing F(ab')z labeled with 18Fand lz51was performed in pairedlabel format, with each animal serving as its own control, a paired t test was used (18). Only results having P < 0.05 have been considered to be statistically significant. RESULTS AND DISCUSSION
Strategies for labeling proteins with 18F must be compatible with the 110-min half-life of this nuclide. In addition, the radiofluorination method should produce a labeled protein which retains the 18F label after in vivo administration. Of critical importance is the ability of the l8F-labe1ed protein to maintain its biological function after labeling. Because of their sensitivity to losses in immunoreactivity as a consequence of radiolabeling procedures, MAb fragments were chosen as model proteins for this study. In addition, there is considerable interest in the clinical application of radiolabeled MAbs, making the development of a method for labeling MAbs with l8F of potential practical utility. Scheme I outlines the four-step approach for labeling MAbs with 18F. Yields for the production of 4-[18F]fluorobenzonitrile, corrected to the end of cyclotron bombardment, were 60-80 7% and comparable to those reported in the literature for the synthesis of this compound by aromatic nucleophilic substitution for the nitro group of 4-nitrobenzonitrile (7, 19). Reduction with lithium aluminum hydride gave 4-[18F]fluorobenzylaminein an end of bombardment overall yield of 65-75%, a range which is considerably higher than that reported previously for the synthesis of 4- [ 18F]fluorobenzylaminefrom [ 18F]fluoride (19).
Garg et al.
Scheme I. Fluorine-18 Labeling of Antibodies
I
l
v
i? ec H , N H / - N r a
Borate bufler pH 8.5
b
Overall yields for the synthesis of [18F]SFBS were between 25 and 40% in a synthesis time of 55-60 min, including HPLC purification. Omitting HPLC purification decreased synthesis time by nearly 20 min, increasing available activity by more than 10%;however, because of difficulties described below with the crude [18F]SFBS preparation, this approach was not pursued after initial investigations. Conjugation efficiencies with [ 18F]SFBS were similar for 81C6 IgG, Mel-14 F(ab')z, antimyosin F(ab')z, and antimyosin Fab, suggesting the versatility of this method. Decay-corrected coupling efficiencies were dependent on protein concentration, increasing from 29 f 3% at 2.5 mg/mL to 32 f 4% at 5.5 mg/mL to 47 f 5% at 8.5 mg/ mL. Other N-succinimidylester radiohalogenation agents exhibit similar concentration-dependent protein labeling (20,211. However, compared to those of [ 18F]SFBS,yields for N-succinimidyl iodobenzoates are higher, and yields with the Bolton-Hunter agent are lower (20,21),suggesting that manipulation of the spacer between the aryl fluoride and the active ester might be an approach for increasing protein-labeling efficiency. Because of the short half-life of 18F, minimizing the incubation time for the ['SFISFBS protein-coupling reaction is critical. Significant differences in proteincoupling efficiency were not observed between 15 and 120 min. For example, in one set of experiments with 500 pg (2.5mg/mL) of antimyosin Fab, coupling efficiencies were 34, 37, 36, 36, and 38% at 15, 30, 60, 90, and 120 min, respectively. On the basis of these results, a 15-20-min reaction period was adopted in order to minimize losses of available I8F-labeled protein due to isotopic decay. The ability to achieve acceptable protein labeling yields in shorter reaction times is an advantage of using SFBS for labeling proteins. In contrast, labeling proteins by reaction with either 3-[18F]fluorobenzimidateor 4- [ 18F]fluorophenacyl bromide appears to require at least 120240 min to obtain reasonable yields (7). Both of these methods also involve prolonged heating at 47 "C. Since the biologic integrity of some proteins may be compromised at elevated temperatures, an additional advantage of SFBS is that the reaction is carried out at 25 "C. Comparison of the current method with those described previously is complicated by the fact that in most cases, reaction time, protein concentration, and the nature of
Bioconjugate Chem., Vol. 2, No. 1, 1991 47
Fluorine-18 Labeling of Antlbodies
I : NO HPLC
40,
O:HPLC
Liver
= 10
Y
Spleen Retention Time
F-18
lB
1
13 minutes
I
2
1
0.5
/I
Hours
Retention 13 minutes Time Figure 1. Size-exclusion HPLC of lsF-labeled antimyosin Fab fragment prepared with (A) crude [18F]SFBS and (B)[18F]SFBS purified by HPLC prior to reaction with Fab fragment. The peak appearing with a retention time of ca. 13 min corresponds to Fab. The shoulder observed in A presumably reflects crosslinked Fab.
the protein are all important variables. With [ 18F]SFBS, about 8-10 mCi of three different 'BF-labeled proteins could be prepared from 100 mCi of [18F]fluoride in a synthesis time of 80-90 min. These yields are more than twice those reported for 18F labeling of human serum albumin using 3-[l8FIfluorobenzimidate(7). The available protein activity per mCi [18F]fluoride for [18F]SFBSis similar to that reported for labeling human serum albumin using 4-['8F]fluorophenacyl bromide. However,when the later reagent was used to label an IgA immunoglobulin, labeling yields were considerably lower, and only about half those seen at similar protein concentrations with SFBS. Despite decreasingpreparation time, omitting the HPLC purification of [18F]SFBSis not recommended. In paired experiments, use of HPLC increased protein coupling yields by 15-40%, presumably by decreasing competition of unlabeled active ester byproducts for reaction with free amino groups on the protein. As shown in Figure 1, sizeexclusion HPLC analysis of 18F-labeled antimyosin Fab indicates that use of crude [18F]SFBSresults in a shoulder on the Fab peak accounting for about 15% of 18Factivity. It is presumed that this reflects cross-linked Fab resulting from exposure to unreacted bifunctional DSS. HPLC purification of ['8F]SFBS also had a significant effect on tissue distribution (Figure 2). With 18F-labeled antimyosin F(ab')n, omitting the HPLC purification of [18F]SFBSsignificantly increased liver and spleen uptake and decreased blood-pool activity at all time points ( P 0.005). For example, at 1h, use of HPLC resulted in more than 2-fold lower liver uptake (6.7 f 0.9 vs 17.1 f 1.4% ID) and nearly 2-fold higher blood-pool activity (20.9 f 1.5 vs 11.6 f 1.3% ID). These differences are consistent with those reported for MAbs labeled with lllIn when methods with and without a propensity for causing protein cross-linking were compared (22, 23). Because of the rapid clearance of MAb F(ab')z and Fab fragments from the blood pool and normal tissues (24-
Figure 2. Tissue distribution of 18F activity in normal mice injected with 1BF-labeled antimyosin Fab. Purification of the [l*F]SFBS intermediate results in significant reduction in liver and spleen uptake and higher activity remaining in blood pool. Table I. Biodistribution of IBF-Labeled Antimyosin Fab Fragment in Normal Mice (HPLC-Purified Intermediate) tissue liver spleen lungs kidneys stomach muscle bone blood brain heart a
0.5 h
% ID/$ lh
2h
5.63 f 0.94 3.65 f 0.52 14.13 f 3.09 17.89 f 3.16 1.44 0.30 0.99 f 0.27 2.08 f 0.34 24.13 f 3.31 0.51 f 0.08 5.24 f 0.97
4.92 f 0.44 2.22 f 0.24 8.62 f 1.97 17.54 f 1.54 1.32 i 0.35 0.97 0.16 0.99 f 0.15 14.89 f 0.94 0.27 f 0.06 3.70 f 0.33
3.04 f 0.26 0.75 f 0.20 5.81 f 0.88 12.48 f 0.94 1.10 0.37 0.64 f 0.11 0.76 f 0.14 8.15 f 0.63 0.02 f 0.02 1.59 f 0.53
*
*
*
Mean f standard deviation; n = 5.
Table 11. Paired-Label Biodistribution of Antimyosin F(ab')z Fragment Labeled with 1*F by Using DSS and IYI by Using N-Succinimidyl 3-[~*~I]Iodobenzoate
tissue
1 h postinjection 18F 1251
2 h postinjection '8F 1251
9.35 f 1.12 7.27 f O.8gb 8.39 f 0.88 6.00 f 0.91b liver 4.53 f 0.36 4.20 f 0.31 3.47 f 0.49 3.33 f 0.26 spleen 17.84 f 5.79 18.18 f 5.93 13.94 f 3.06 14.29 f 2.95 lungs kidneys 13.99 f 1.25 11.79 f 0.62b 15.55 f 1.32 9.95 f 0.86b 2.02 f 0.30 2.21 f 0.31 stomach 1.68 f 0.76 1.83 f 0.81 0.80 f 0.07 0.86 f 0.09 0.73 f 0.09 0.80 f 0.08 muscle 1.69 f 0.30 1.85 f 0.17 1.40 f 0.18 1.96 f 0.18b bone 32.09 f 1.94 31.80 2.00 27.48 f 0.99 27.44 f 1.12 blood 0.62 f 0.10 0.65 f 0.05 0.71 f 0.05 0.76 f 0.05 brain 5.55 f 0.67 6.12 f 0.68 6.33 f 0.47 6.66 f 0.49 heart a Mean f standard deviation; n = 5. Significance of difference P < 0.01 determined by two-sided paired t test; all other differences not significant.
*
26),their pharmacokinetics appear to be more compatible with the 110-min half-life of 18F. The tissue distribution of 18F-labeled antimyosin Fab in normal mice at 0.5-2 h is summarized in Table I. Fluorine-18 activity cleared rapidly from the blood pool, reaching a level of 8.15 f 0.63 5% ID/g a t 2 h. After 0.5 h, Fab uptake in the kidneys was higher than that observed in other tissues. Although little data at early time points is available for comparison,
48
Garg et al.
Bioconjugate Chem., Vol. 2, No. 1, 1991
the tissue distribution of 18F-labeled antimyosin Fab is similar to that reported for other Fab fragments radioiodinated by using the Iodogen method (25,26). A paired-label biodistribution study was performed in normal mice to compare the pharmacokinetics of antimyosin F(ab’)2labeled by reaction with [18F]SFBSandN-succinimidyl 3- [ 1251]iodobenzoate. This radioiodination method was used because it has been shown to decrease the dehalogenation of MAbs and F(ab’)2 fragments in vivo (27,28). As summarized in Table 11, in most tissues the uptake of ‘8F and 1251 was nearly identical at 1 and 2 h. Small but significant differences were observed in the liver a t both time points and in the bone a t 2 h. Increased liver uptake of 18Fmay be related to a small degree of crosslinking. However, the data presented in Figure 2 would suggest that if this were a major factor, differences in spleenic and blood-pool uptake of 18Fand 1251 would also be seen, and this was not the case. The most significant difference between ‘8F and lZ5Iactivity was noted in the kidneys where 19-56% higher levels of I8Fwere observed. Complementary differences in bladder activity were seen (125I,17.8f 1.9% ID;18F,ll,9f 1.4% IDat2h),suggesting that the excretion rates of l8F- and lZ5I-labeledFab (or their labeled catabolites) are slightly different. Of critical importance is the demonstration that a protein-labeling method does not destroy the immunoreactivity of the MAb or MAb fragment. Although methods for labeling proteins with 18Fhave been described previously (7-9), retention of immunocompetence of a MAb or MAb fragment after radiofluorination has not been reported. Initially, the effect of HPLC purification of the [18F]SFBS acylation agent on the binding of l8Flabeled antimyosin Fab was investigated with a myosin affinity column. In two paired preparations, immunoreactivity with HPLC-purified [18F]SFBSwas 82 and 84 % , compared with 49 and 67 7% when crude [ 18F]SFBS was used. Immunoreactivity of 11 batches of 18F-labeledantimyosin Fab using HPLC-purified [18F]SFBSwas 75 f 9%. Binding of ‘8F-labeled antimyosin F(ab’)2 to the myosin affinity column was 89 f 5% compared to less than 570 for 18F-labeledcontrol Mel-14 F(ab’)a. The higher immunoreactivity of divalent F(ab’)z compared to monovalent Fab is consistent with the results reported for other MAbs (24,25,29). No published data are available for the binding of antimyosin fragments to myosin affinity columns after other radiolabeling procedures. Nevertheless, the immunoreactivities measured for lsF-labeled antimyosin Fab and F(ab‘)z compare favorably with those reported in the literature for other radiolabeled MAb fragments (24-26, 29). For example, the binding of 1251-labeledanti-CEA MAb fragments to carcinoembyronic antigen affinity columns ranged between 50 and 65 % for two Fab fragments and 54-77 7% for four F(ab’)2 fragments (29). In summary, we have described a method for labeling proteins with ‘8F in good yield a t specific activities of up to 11 mCi/mg in a synthesis time of about 80-90 min, nearly 1 h shorter than previously published methods. HPLC purification of the [‘8F]SFBS intermediate is required to achieve optimal protein coupling efficiency, in vivo tissue distribution, and immunoreactivity of l8Flabeled MAb fragments. The results from the immunoreactivity and in vivo stability measurements suggest that [18F]SFBSmay be a valuable reagent for labeling MAbs and their fragments with 18Ffor use with PET. To this end, we have initiated an investigation (to be reported in a subsequent publication) of the pharmacokinetics of 18Flabeled antimyosin F(ab’)z and Fab in a canine myocar-
dial infarct model. In five animals studied to date, 18Flabeled MAb fragments were observed to selectively accumulate in myosin-rich infarcted myocardium. ACKNOWLEDGMENT
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