Evaluation of iodovinyl antibody conjugates - American Chemical

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Bioconjugafe Chem. 1990, I , 154-161

Evaluation of Iodovinyl Antibody Conjugates: Comparison with a p-Iodobenzoyl Conjugate and Direct Radioiodination Stephen W. Hadley* and D. Scott Wilbur NeoRx Corporation, 410 W. Harrison Street, Seattle, Washington 98119. Received January 29, 1989

The preparations and conjugations of 2,3,5,6-tetrafluorophenyl5-[ 1251/1311]iodo-4-penten~ate (7a) and 2,3,5,6-tetrafluorophenyl3,3-dimethyl-5-[ '251/1311]iodo-4-pentenoate (7b) to monoclonal antibodies are reported. Reagents 7a and 7b were prepared in high radiochemical yield by iododestannylation of their corresponding 5-tri-n-butylstannyl precursors. Radioiodinated antibody conjugates were prepared by reaction of 7a or 7b with the protein at basic pH. Evaluation of these conjugates by several in vitro procedures demonstrated that the radiolabel was attached to the antibody in a stable manner and that the conjugates maintained immunoreactivity. Comparative dual-isotope biodistribution studies of a monoclonal antibody Fab fragment conjugate of 7a and 7b with the same Fab fragment labeled with N-succinimidyl p-[1311]iodobenzoate(PIB, p-iodobenzoate, 2) or directly radioiodinated have been carried out in tumor-bearing nude mice. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 2 demonstrated that the biodistributions were similar in most organs, except the neck tissue (thyroid-containing) and the stomach, which contained substantially increased levels of the 7a label. Coinjection of the Fab conjugate of 7a with the Fab fragment radioiodinated by using the chloramine-?' method demonstrated that the biodistributions were remarkably similar, suggesting roughly equivalent in vivo deiodination of these labeled antibody fragments. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 7b indicated that there was approximately a 2-fold reduction in the amount of in vivo deiodination of the 7b conjugate as compared to the 7a conjugate.

Radioiodinated monoclonal antibodies are being investigated for their application to the imaging and therapy of cancer (1-4). The standard method for producing radioiodinated monoclonal antibodies is direct attachment of the radioiodine to the protein. Direct protein radioiodination methods have been extensively studied (5). Generally, these methods employ oxidants, such as chloramine-T or Iodogen, for the generation of electrophilic iodine species which react principally with the activated aromatic ring of tyrosine amino acids of the protein (6). However, a significant problem affecting the diagnostic and therapeutic potential of directly radioiodinated antibodies is their susceptibility to in vivo deiodination. Presumably in vivo deiodinases are capable of recognizing the structural similarity between the thyroid hormones and iodinated tyrosines of the antibody or its catabolized fragments (7). Deiodination results in significant, undesirable accumulation of radioiodine in both the thyroid and stomach tissues. More important, however, is the potential for loss of radioactivity from that localized at tumors. The in vivo instability of directly labeled antibodies has prompted the development of alternate methods of radioiodination which resist in vivo deiodination. For example, we have developed a protein radioiodination reagent, N-succinimidyl p-iodobenzoate (PIB, p-iodobenzoate, 2), which employes a nonactivated, nonphenolic iodophenyl group to attach the radioiodine to the antibody in a stable manner (8). The PIB reagent (2) can be conveniently prepared in high specific activity by the radioiododestannylation of N-succinimidyl p-(tri-nbutylstanny1)benzoate (l),as shown in Scheme I. The PIB reagent (2) is then conjugated with antibody by acylation of the t amino groups of the lysines on the surface of the antibody. In a similar manner, Zalutsky has reported N-succinimidyl m-(tri-n-butylstanny1)benzoate

Scheme I 0

0

NCS/N~*I ___)

I

SnBu3 1

2

(ATE, alkyl tin ester) as a protein radioiodination reagent (9). Antibodies labeled with either PIB (2) or ATE have been shown to undergo negligible, if any, in vivo deiodination. Vinyl iodides are comparable to aryl iodides in terms of their chemical and biological stabilities and they are more stable than aliphatic iodides (10). Thus, a reasonable alternative to an iodophenyl group for the attachment of radioiodine to antibodies in a stable manner is an iodovinyl group. Indeed, iodovinyl moieties have been used in the development of new radiopharmaceuticals. For example, iodovinyl-substituted fatty acids have been studied for myocardial imaging (II ) , iodovinyl estradiols have been used to detect hormone dependent tumors (12),and iodovinyl glucose has been investigated for use in evaluating brain disorders (13). Reported herein are the results of an investigation involving two new protein radioiodination reagents: 2,3,5,6-tetrafluorophenyl 5iodo-4-pentenoate (7a) and 2,3,5,6-tetrafluoropheny13,3dimethyl-5-iodo-4-pentenoate (7b). In this investigation, the synthesis and radioiodination reactions used to prepare these reagents were studied. Evaluation of the radioiodination reagents included biodistribution studies comparing a Fab fragment labeled with 2, 7a, or 7b, or directly labeled by the chloramine-T method. The

1043-1802/90/2901-0154$02.50/00 1990 American Chemical Society

Iodovinyl Antibody Conjugates

results obtained from the biodistribution studies have allowed comparison of the different radioiodination methods with respect to in vivo deiodination and tumor localization. EXPERIMENTAL PROCEDURES

General Procedures. All compounds gave spectra in accord with their proposed structures. NMR spectra were obtained in CDCl, solution with a Varian Gemini-200 200-MHz instrument. The proton chemical shifts (6) are reported in ppm downfield from internal Me,Si (0.00 ppm). The carbon chemical shifts (6) are reported in ppm downfield from Me,Si; 6(Me,Si) = G(CDC1,) - 77.0 ppm. IR spectra were obtained on a Perkin-Elmer 1310 infrared spectrophotometer. Mass spectral data were obtained on a VG 7070H instrument operating in the E1 mode or a VG 70SEQ instrument operating in the FAB mode. In general, all reagents were reagent grade or better and were used as purchased. Toluene (anhydrous), T H F (anhydrous), 4-pentynoic acid, 2,3,5,6-tetrafluorophenol, N,N'-dicyclohexylcarbodiimide,Et,B, N-chlorosuccinimide, and Diazald were purchased from Aldrich Chemical Co. Bu,SnH was purchased from Alfa Products and distilled prior to use. HPLC solvents were obtained as HPLC grade and were filtered (0.2 pm) prior to use. Phosphate-buffered saline (PBS) was purchased from Gibco Labs as Dulbecco's phosphate-buffered saline (#3104190). Radioiodine was obtained from Du Pont/NEN (North Billerica, MA). Iodine-125 was obtained as a NalZ5Isolution in 0.1 N NaOH (high concentration) at 17 Ci/mg in 2- and 5-mCi quantities. Iodine-131 was obtained as a Na1311 solution in 0.1 N NaOH (high concentration) at 7-12 Ci/mg in 5-mCi quantities. Flash chromatography was performed with EM Science silica gel 60, 230-400 mesh. HPLC was conducted with Beckman Model llOB pumps, a Beckman Model 153 UV detector, a Beckman Model 170 radioisotope detector, and a Rheodyne Model 7125 injector. Spectrophotometric analyses were performed by UV detection at 254 nm and radiometric analyses were achieved by NaI scintillation optimized to the radionuclide used. Integration and plotting were accomplished by either a n integrator/plotter (Hewlett-Packard, Model 3390) or a computer with a chromatographic software package (Dynamic Solutions, Maxima 820 workstation). Reversephase HPLC chromatography was run on a Whatman Partisphere C-18 column, 4.6 mm X 12.5 cm, using a gradient solvent system operating at 1.0 mL/min. Solvent A in the gradient was 98% MeOH/2% H,O (H,O contained 1% HOAc). Solvent B was 10% MeOH/9O% H,O (H,O contained 1%HOAc). The gradient began a t 25% B. After 3 min the gradient was decreased in percent B over the next 7 min to 2 % B and was held there for 10 min. With this gradient the retention times for the compounds were as follows: 6a (20.0 min), 7a (5.3, 5.6 min), 6b (21.2 min), 7b (9.6 min), and iodide (1.4 min, solvent front). Size-exclusion HPLC was performed on a Zorbax Bio-Series GF-250, 9.4 mm X 24 cm, column (Du Pont) eluted with 0.2 M sodium phosphate, pH 6.8, a t a flow rate of 1.0 mL/min. Radiochemical purities of labeled proteins were determined by "instant" thin-layer chromatography (ITLC). Silica gel impregnated glass fiber strips (ITLC-Gelman) were activated by heating a t 110 "C for 30 min prior to use. After elution, the plates were cut into small strips (horizontal) and were counted in a gamma counter (Packard Autogamma 5650) to detect radioactivity. Iodine-

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125 samples were counted in plastic tubes with a 15-80 keV window, whereas iodine-131 samples were counted with a 260-470 keV window. Radiochemical purity, as determined by ITLC, was expressed as the percent of counts (cpm) on the bottom half (origin) of the strip divided by the total counts on the strip. Synthesis of Methyl 4-Pentynoate (4a) and Methyl 3,3-Dimethyl-4-pentynoate(4b). To a solution of 4pentynoic acid (3a, 510 mg, 5.2 mmol) in ether (7.5 mL) at 0 "C was added an ether solution of CH,N, (14) until the faint yellow color of excess CH,N, persisted. The reaction was stirred for 30 min, then the excess CH,N, was quenched by the addition of acetic acid. The solution was extracted with saturated NaHCO, (2 X 10 mL), washed with saturated NaCl(1 X 10 mL), and dried over MgSO,. The solution was filtered and the ether was removed by simple distillation at atmospheric pressure to afford 4a (545 mg, 94%) as a light yellow liquid: 'H NMR 6 3.72 (s, 3 H), 2.55 (m, 4 H), 1.99 (t, J = 2.5 Hz, 1 H) . In a similar manner, 4b was prepared from 3,3-dimethyl-4-pentynoic acid (3b) (15) (344 mg, 2.73 mmol) in 99% yield: 'H NMR 6 3.70 (s, 2 H), 2.17 (s, 1 H), 1.36 (s, 6 HI. Synthesis of Methyl 5-(Tri-n-Butylstanny1)-4pentenoate (5a). To a solution of 4a (168 mg, 1.5 mmol) in anhydrous toluene (3.0 mL) under N, a t 0 "C was added Bu,SnH (0.40 mL, 1.5 mmol) followed by Et,B (0.15 mL, 1.0 M solution in hexanes, 0.15 mmol) (16). After 3 h at 0 "C additional Bu,SnH (0.20 mL, 0.75 mmol) was added. The resulting solution was allowed to warm to room temperature and stirred overnight. The solvents were evaporated under reduced pressure and the resulting oil was purified by flash chromatography (25 X 150 mm), eluting with a step gradient of 100% hexanes, 5% EtOAc/ hexanes, 10% EtOAc/hexanes, to afford 5a (264 mg, 44%) as a 50/50 mixture of cis/trans isomers. 'H NMR 6 6.47 (d of t, J = 12, 7 Hz, 0.5 H), 6.16-5.64 (m, 1.5 H), 3.69 (s, 1.5 H), 3.67 (s, 1.5 H), 2.60-2.25 (m, 4 H), 1.80-1.15 (m, 18 H), 0.92 (t, J = 7 Hz, 4.5 H), 0.89 (t, J = 7 Hz, 4.5 H); I3C NMR 6 173.87, 173.52, 146.86, 146.61, 130.20, 128.94, 51.36,51.30,34.13, 33.26,32.49,31.96,29.01,28.90, 27.11, 27.06, 13.44, 9.98, 9.16. Synthesis of 2,3,5,6-Tetrafluorophenyl 5-(Tri-nbutylstannyl)-4-pentenoate(sa). To a solution of 5a (140 mg, 0.35 mmol) in EtOH (1.0 mL) was added a solution of KOH (208 mg, 3.7 mmol) in EtOH (1.0 mL), followed by H,O (0.20 mL). The resulting solution was stirred at room temperature for 2 h. The solution volume was reduced to one-half the original volume by evaporation under reduced pressure. The residue was suspended in ether (10 mL) and acidified with 1.0 N HC1 (4.0 mL, 4.0 mmol). The aqueous phase was separated and extracted with ether (2 X 5 mL). The ether layers were combined, washed with saturated NaCl (1 X 5 mL), and dried over MgSO,. The solution was filtered and the solvent was evaporated under reduced pressure to afford the crude acid as an oil. The crude acid was dissolved in anhydrous T H F (3.0 mL), and 2,3,5,6-tetrafluorophenol(80 mg, 0.48 mmol) in THF (0.5 mL) was added, followed by N,N'-dicyclohexylcarbodiimide (101 mg, 0.49 mmol). The solution was stirred overnight at room temperature, during which time a white precipitate formed. The T H F was evaporated under reduced pressure, CH,CN was added, and the solution was filtered. The filtrate was evaporated under reduced pressure to afford an oil. The crude product was purified by flash chromatography (10 X 170 mm), eluting with 5% EtOAc/hexanes, to afford

156


6a (118 mg, 63%) as a 50/50 mixture of &/trans isomers: 'H NMR 6 6.99 (m, 1 H), 6.54 (d o f t , J = 13, 7 Hz, 0.5 H), 6.24-5.76 (m, 1.5 H), 2.87-2.40 (m, 4 H), 1.701.15 (m, 18 H), 0.92 (t, J = 7 Hz, 4.5 H), 0.88 (t, J = 7 Hz, 4.5 H); 13C NMR 6 169.29, 169.04, 148.79 (m), 148.70 (m), 145.48, 145.34, 143.72 (m), 143.63 (m), 138.44 (m), 138.24 (m), 131.70, 130.33, 103.24 (t, J = 23 Hz), 103.17 (t, J = 23 Hz), 33.59, 32.68, 32.15, 31.75, 29.08, 28.96, 27.16, 13.49, 10.07, 9.26; IR (neat, thin film) 3090, 2960, 2920,2870,2850,1790,1645,1600,1530,1490,1180,1100, 1080, 960 cm-'; MS (FAB) m / e 539 (M + 1, '"Sn), 537 (M + 1, "'Sn), 481 (M + 1, loss of butane, '"Sn), 479 (M + 1, loss of butane, l18Sn) amu. Synthesis of Methyl 3,3-Dimethyl-5-(tri-n-butylstannyl)-4-pentenoate (5b). To a solution of 4b (123 mg, 0.88 mmol) in anhydrous toluene (3.5 mL) under N, at 0 "C was added Bu,SnH (0.30 mL, 0.95 mmol), followed by Et,B (0.10 mL, 1.0 M solution in hexanes, 0.10 mmol). The resulting solution was allowed to warm to room temperature and stirred overnight. The solvents were evaporated under reduced pressure and the resulting oil was purified by flash chromatography (25 X 150 mm), eluting with a step gradient of 100% hexanes, 5 % EtOAc/hexanes, 10% EtOAc/hexanes, to afford 5b (262 mg, 69%) as an oil: 'H NMR 6 5.92 (AB, AvAB = 11.8 Hz, J = 10 Hz, 2 H), 3.62 (s, 3 H), 2.30 (s, 2 H), 1.601.20 (m, 18 H), 1.12 (s, 6 H), 0.89 (m, 9 H); 13C NMR 6 172.57, 156.83, 122.47, 50.92, 46.50, 38.15, 28.88, 27.04, 26.75, 13.47, 9.23.

Synthesis of 2,3,5,6-Tetrafluorophenyl 3,3-Dimethyl-5-(tri-n-butylstannyl)-4-pentenoate (6b). To a solution of 5b (150 mg, 0.35 mmol) in EtOH (2.0 mL) was added a solution of KOH (250 mg, 4.46 mmol), followed by H,O (0.20 mL). The resulting solution was stirred at room temperature for 24 h. The solution volume was reduced to one-half the original volume by evaporation under reduced pressure. The residue was suspended in ether (10 mL) and acidified with 1.0 N HC1 (4.5 mL, 4.5 mmol). The aqueous phase was separated and extracted with ether (2 X 5 mL). The ether layers were combined, washed with saturated NaCl ( 1 X 5 mL), and dried over MgSO,. The solution was filtered and the solvent was evaporated under reduced pressure to afford the crude acid as an oil. The crude acid was dissolved in anhydrous T H F (3.0 mL), and 2,3,5,64etrafluorophenol (87 mg, 0.52 mmol) in T H F (0.5 mL) was added, followed by N,N'-dicyclohexylcarbodiimide(107 mg, 0.52 mmol). The solution was stirred overnight at room temperature, during which time a white precipitate formed. The T H F was evaporated under reduced pressure, CH,CN was added and the solution was filtered. The filtrate was evaporated under reduced pressure to afford an oil. The crude product was purified by flash chromatography (10 X 170 mm), eluting with 5 % EtOAc/hexanes, to afford 6b (125 mg, 69%) as an oil: 'H NMR 6 6.97 (m, 1 H), 5.99 (AB, AuAB = 12.3 Hz, J = 19 Hz, 2 H), 2.64 (s, 2 H), 1.6-1.2 (m, 18 H), 1.22 (s, 6 H), 0.85 (t, J = 7 Hz, 9 H); 13C NMR 6 167.71, 155.65, 148.76 (m), 143.72 (m),138.51 (m), 123.79, 103.06 (t, J = 23 Hz), 45.69, 38.41, 28.89, 27.06, 26.70, 13.45, 9.24; IR (neat, thin film) 3080, 2960, 2920, 2870, 2850,1790,1645, 1595,1525,1490,1180,1090,960 cm-'; MS (FAB) m / e 567 (M + 1, '"Sn), 565 (M + 1, '"Sn), 509 (M + 1, loss of butane, lZ0Sn),507 (M + 1, loss of butane, "'Sn) amu. Synthesis of 2,3,5,6-Tetrafluorophenyl 5-Iodo-4pentenoate (7a) and 2,3,5,6-Tetrafluorophenyl3,3Dimethyl-5-iodo-4-pentenoate (7b). To a solution of 6a (14 mg, 26 pmol) in CDCl, (0.4 mL) was added a solu-

Hadley and Wilbur

tion of IC1 (0.23 mL, 0.11 M solution in CDCl,, 25 pmol) until the faint purple color of excess IC1 persisted. The crude product was purified by flash chromatography (5 X 80 mm), eluting with 5% EtOAc/hexanes, to afford 7a (6.4 mg, 66%) as a 50/50 mixture of &/trans isomers: 'H NMR 6 7.01 (m, 1 H), 6.59 (d o f t , J = 15, 7 Hz, 0.5 H), 6.42-6.08 (m, 1.5 H), 2.95-2.70 (m, 2 H), 2.702.45 (m, 2 H); MS (EI) m / e 374 (M+), 247,209,181,167 amu. In a similar manner 7b was prepared from 6b (19 mg, 35 pmol) in 74% yield: 'H NMR 6 7.01 (m, 1 H), 6.69 (d, J = 15 Hz, 1 H), 6.20 (d, J = 15 Hz, 1H), 2.66 (s, 2 H), 1.25 (s, 6 H); MS (EI) m / e 402 (M+), 275, 237, 209, 195 amu. Synthesis of 2,3,5,6-Tetrafluorophenyl 5-[lZ5I/ 1311]Iodo-4-pentenoate(7a) and 2,3,5,6-Tetrafluorophenyl 3,3-Dimethyl-5-[ lZ5I/1311]iodo-4-pentenoate (7b). Into a reaction vial fitted with a septum seal was placed a 25-pL solution containing either 6a or 6b (25 pg, 0.050 pmol) in 5% HOAc/MeOH, a 10-pL solution containing N-chlorosuccinimide (10 pg, 0.075 pmol) in MeOH, and 10 pL of PBS. To this mixture was added the desired quantity of NalZ5Ior Na1311 (up to 5 mCi, volume not to exceed 10 pL if diluted with PBS). After 5 min at room temperature the reaction was quenched by the addition of a 10-pL solution of NaHSO, (0.72 mg/ mL Na,S,O, in H,O, 0.075 pmol of NaHSO,). An aliquot was removed for HPLC analysis. Following this procedure, 7a was prepared from 6a and NalZ5I (2 pL, 106 pCi) in 88% radiochemical yield as a 45/55 mixture of cis/trans isomers. Likewise, 7b was prepared from 6b and NalZ5I(2 pL, 112 pCi) in 91% radiochemical yield as a single isomer. The MeOH was carefully evaporated from the reaction mixture by passing a stream of N, gas through the reaction vial. To trap any volatile radioiodide, the N, gas was introduced into the vial by means of a needle inlet, and the vial was vented with a needle outlet attached to a 3 mL syringe barrel filled with granulated charcoal. The evaporated product was used without purification in the subsequent antibody conjugation reaction. Preparation of NR-ML-05 Fab Labeled with [ lZ5I]7a.To a vial containing [lZ5I]7a(850 pCi) was added a solution of NR-ML-05 Fab (500 pg in 56 pL PBS) in NaHCO,/Na,CO, buffer (100 pL, 1.0 M, pH 9.2). The reaction was incubated at 37 "C for 30 min and then quenched by the addition of a glycine solution (10 pL, 1 M glycine in 1.0 M NaHCO,/Na,CO,, pH 9.2). The conjugate was purified with a Pharmacia PD-10 column eluted with PBS to afford 322 pCi (38%) of labeled NR-ML-05 Fab. The purity, as assessed by ITLC, was 98.7% and the specific activity was 0.73 pCi/pg. Preparation of NR-ML-05 Fab labeled with [l3l1]7a or [lZ5I]7bwas accomplished as described above. Preparation of NR-ML-05 Fab labeled with 2 is described elsewhere (8). Direct Radioiodination of NR-ML-05Fab. To a vial containing Nalz5I (641 pCi), PBS (100 pL), and NR-ML05 Fab (500 pg in 56 p L PBS) was added a solution of chloramine-T (10 pL of a 1.0 mg/mL solution in H,O). After 5 min at room temperature the iodination reaction was quenched by the addition of NaHSO, (20 pL, 0.72 mg/mL Na,S,05 in H,O). The crude, labeled material was immediately purified with a Pharmacia PD-10 column eluted with PBS to afford 496 pCi (77%) of labeled NR-ML-05 Fab. The purity, as assessed by ITLC, was 99.4% and the specific activity was 0.98 pCi/pg.

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Iodovlnyl Antibody Conjugates

Chemical Challenge Experiments. To test tubes containing 1.0 mL of either 12% TCA, 6 M urea, 10 mM dithiothreitol (DTT), 1.0 M (pH 9.2) sodium carbonate buffer, 95% ethanol, 1 M glycine in 1.0 M (pH 9.2) carbonate buffer, or PBS (control) was added 25 pg of radiolabeled antibody. The samples were lightly vortexed and an aliquot was removed and analyzed by ITLC (80% MeOH) as the t = 0 time point. The tubes were then incubated a t 37 "C for 24 h. Aliquots were periodically removed and analyzed by ITLC. Serum Stability. Samples of freshly isolated human serum were filtered through 0.2-pm filters and placed into sterile, capped culture tubes. Twenty-five micrograms of radioiodinated antibody were added to each 0.5-mL serum sample. Duplicate samples were lightly vortexed and an aliquot was removed and analyzed by ITLC (80% MeOH) as the t = 0 time point. The tubes were then incubated a t 37 "C for several days. Aliquots were periodically removed and analyzed by ITLC. Immunoreactivity Assessment. The immunoreactivities of the radioiodinated antibodies were determined from a cell-binding assay using a fixed labeledantibody concentration and a varied number of antigen positive cells (17). Duplicate 110-pL aliquots of 25, 20, 15, and 10 X lo6 cells/mL were added to eight 0.5-mL tubes containing 55 pL of assay buffer (PBS/11% BSA/ 0.1% sodium azide). T o a ninth tube containing 55 pL of assay buffer was added 110 pL of the 25 X lo6 cells/ mL preparation, followed by 55 pL of a 200 pg/mL solution of unlabeled antibody. The tubes were mixed and incubated at 4 OC for 1 h. Then 55 pL of a 160 ng/mL solution of radioiodinated antibody was added to each of the nine tubes. The tubes were incubated with mixing at 4 "C for 2 h. Aliquots (200 pL) of the labeled antibody/ cell reaction mixture were withdrawn from the tubes and overlaid onto an oil mixture (18). The overlaid oil tubes were cooled to 4 OC, centrifuged for 30 s, and cut through the oil layer to separate the cell pellet from the aqueous layer. The cut tubes were placed into 12 X 75 mm glass tubes and counted in a gamma counter. The percent cell-bound radioactivity was calculated by dividing the counts associated with the cell pellet by the total counts. Duplicates were averaged and the percent nonspecifically bound radioactivity determined from the ninth tube was subtracted to give the percent specific binding. Adjustments to the percent specific binding were made according to the ITLC purities. Average value obtained for immunoreactivity of NRML-05 Fab labeled with PIB (2) is 72 f 11% ( n = 50). The measured immunoreactivity of NR-ML-05 Fab labeled with 7a or 7b or directly labeled by using the chloramine-T method used in animal biodistribution studies described herein was within these bounds. Biodistribution Studies. Female athymic nude mice (nu/nu) were obtained from Simonsen Laboratories, Inc., Gilroy, CA, and were used a t 10-12 weeks of age. Tumor xenografts were produced by subcutaneous implantation of 2.5 X lo6 A375 Met Mix cultured human melanoma tumor cells obtained from American Type Culture Collection, Rockville, MD (item no. ATCC CRL 1619). The tumor cells were grown for 7-10 days before use. Biodistribution studies employed NR-ML-05 Fab labeled with 7a, 7b, or 2 or by the chloramine-T method, using either iodine-125 or iodine-131. NR-ML-05 is a murine IgG 2b monoclonal antibody which recognizes a 250-kDa proteoglycan antigen expressed on melanoma cells. Purified radioiodinated conjugates were coinjected into mice such that each animal received a 5-pg dose of each radi-

olabeled preparation and a total of 10 pg of injected protein. Mice were injected intravenously via the lateral tail vein and sacrificed by cervical dislocation at prescribed times postinjection. Blood samples were obtained by retroorbital bleeding. Syringes were weighed before and after injection to determine the volume of conjugate injected. The activity per unit volume was calculated from standards. The animals were weighed (23.34 f 1.61 g, n = 24) and marked for identification in all experiments. Twelve different tissues were evaluated: blood, tail (injection site), tumor, skin, muscle (thighs), lung, liver, spleen, stomach, neck including thyroid (the major portion of the muscle from the front of the neck), kidney (both), and intestine. The excised tissues were blotted, weighed, and counted in a dual-channel gamma counter. The raw counts were collected, and the contribution of iodine131 counts to the iodine-125 window (spillover), as determined by iodine-131 standards, was subtracted from the iodine-125 counts and decay corrected. Summarized data include mean f SD of percent injected dose per gram, tissue-to-blood ratios, and tumor-to-tissue ratios. RESULTS

Chemistry. The first reagent selected for evaluation (7a).The was 2,3,5,6-tetrafluorophenyl5-iodo-4-pentenoate design of this reagent was based on the stability of the iodine-carbon bond, which is influenced by the electronic nature of the carbon-carbon double bond. Since acylation chemistry was to be used to conjugate the reagent to antibody, it was deemed necessary to isolate the iodovinyl group from the acyl group by a minimum of two intervening saturated carbon atoms. In such a manner the electronic influence of the carbonyl group on the iodovinyl group would be minimized. In addition, reasoning that the placement of a quaternary carbon atom p to the carbonyl group and adjacent to the iodovinyl group might impart even greater stability to the iodine-carbon bond, we evaluated 2,3,5,6-tetrafluorophenyl3,3-dimethyl-5iodo-4-pentenoate (7b). The method of choice to prepare radioiodinated compounds in high specific activity is to use organometallic reagents. Vinylboronic acids (19, 20), vinylmercurials (21, 221, and vinylstannanes (12) have been used to prepare the corresponding radioiodinated iodovinyl compounds, typically in high yield. Vinylstannanes were chosen for these studies since they can be conveniently prepared by hydrostannylation of the corresponding acetylenic compounds under relatively mild conditions. In addition, water-insoluble vinylstannanes would be less prone to conjugate to antibody, an advantage because it was desirable that the iodovinyl reagents not be purified from the vinyltin precursors prior to reaction with antibody. Avoiding purification of the iodovinyl reagents would simplify the labeling procedure and minimize handling of radioiodinated compounds. Vinyltin active esters 6a and 6b were synthesized from the corresponding acetylenic carboxylic acids 3a and 3b as shown in Scheme 2. Treatment of 3a or 3b with excess CH,N, afforded the methyl esters 4a or 4b. Introduction of a tri-n-butylstannyl group was accomplished by reaction of esters 4a or 4b with Bu,SnH catalyzed by Et,B (16) to afford 5a in 44% yield or 5b in 69% yield. Ester 5a was shown by 'H and I3C NMR to be a 50/50 mixture of cis and trans isomers, while ester 5b was shown to be a single isomer, presumably trans. Conversion of methyl ester 5a or 5b into its corresponding 2,3,5,6-tetrafluorophenyl (TFP) ester was accomplished by basecatalyzed hydrolysis followed by reaction of the interme-

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Hadley and Wilbur

Scheme I1

3a:R=H 4a:R=H b:R=Me b:R=Me Bu3Sn / Bu,Sn 0 T C O 2 C H 3 y C O z T F P R R R R 5a: R = H Ga:R=H

-

b:R=Me

-

b:R=Me

'I

tion prior to purification of the crude conjugate by gel filtration. Radiochemical purities of the various iodovinyl conjugates were routinely in excess of 98.0%, as assessed by ITLC. Omission of the glycine quench afforded consistently lower radiochemical purities of the purified conjugates. To improve the yield and kinetics of the conjugation reaction, we attempted to prepare the more reactive Nhydroxysuccinimide (NHS) ester of 7a. While the corresponding NHS ester of the vinyltin TFP ester 6a was successfully prepared, the NHS ester group proved to be too susceptible toward hydrolysis to survive the radioiodination conditions, and only the radioiodinated carboxylic acid was obtained. In Vitro Analyses. Purified iodovinyl conjugates were analyzed by HPLC with a size-exclusion column. The column effluent was monitored by in-line UV and gamma detectors. The UV peak and the gamma peak coeluted, demonstrating that the radioactivity was associated with the protein. Furthermore, there was no apparent damage to the protein as a result, of the radiolabeling, since there was an absence of high molecular weight aggregates, low molecular weight fragments, and peak tailing in the HPLC traces. Indeed, the HPLC profiles of the iodovinyl conjugates were indistinguishable from the HPLC profiles of unlabeled antibody and antibody labeled with PIB (2) or antibody directly labeled with chloramine-?'. The iodovinyl conjugates were analyzed by SDSPAGE, reduced and nonreduced, combined with autoradiography. The SDS-PAGE protein bands corresponding to the iodovinyl conjugates were superimposable onto bands corresponding to antibody controls. Also, no additional bands were present in the iodovinyl lanes as compared to those of the controls. Autoradiography demonstrated that the radiolabel comigrated with the protein bands on the gel and that all radiolabel was associated with a protein band. Chemical challenge studies were performed to evaluate whether unreacted iodovinyl reagents 7a and 7b or their corresponding carboxylic acids were nonspecifically associated with antibody or were bonded to a group other than an amine on the protein. In those experiments, the purified iodovinyl conjugates were exposed to a variety of chemical conditions, such as 12% TCA, 6 M urea, 95% ethanol, 10 mM dithiothreitol, 1 M glycine, and 1M carbonate buffer, pH 9.2. The results demonstrated that the postchallenge parities were essentially equivalent to the prechallenge purities, suggesting that the radioiodine was covalently attached to the antibody in a stable manner. The serum stabilities of the iodovinyl conjugates were also evaluated. Incubation of the conjugates at 37 "C in freshly prepared human serum demonstrated that there was no appreciable decrease in radioactivity associated with the antibody over several days. The immunoreactivity of the iodovinyl conjugates was routinely determined prior to in vivo evaluation. In all cases, the immunoreactivity of the iodovinyl conjugates, as measured by a radiolabeled cell binding assay, was equivalent to the immunoreactivity of PIB (2) and chloramine-T-labeled controls. Biodistribution Studies. To evaluate the stability of iodovinyl conjugates with respect to in vivo deiodination and tumor localization, several biodistribution studies were performed. The antibody selected for these studies was the Fab fragment of NR-ML-05, a murine antimelanoma antibody that recognizes a 250-kDa proteoglycan antigen expressed on melanoma cells. The biodistribu-

TCO,TFP 0 R R 7a: R = H

b:R=Me

diate carboxylic acid with 2,3,5,6-tetrafluorophenol mediated by N,N'-dicyclohexylcarbodiimideto afford 6a or 6b in 63% or 69% yield, respectively. Hydrostannylation of acids 3a and 3b was also investigated as a more direct approach to obtain the desired vinyltin active esters 6a and 6b. Unfortunately, reaction of 3a or 3b with excess Bu3SnH in the presence of AIBN (23) or Et3B afforded vinyltin-containing carboxylic acid derivatives, which proved more difficult to purify and characterize. Authentic standards were synthesized to confirm the identities of radioiodinated compounds. Thus, vinyltin active esters 6a and 6b were treated with IC1 in chloroform to provide iodovinyl active esters 7a and 7b in 66% and 74% yield, respectively. Simple iodovinyl active ester 7a was shown by NMR to be a 50150 mixture of cis and trans isomers. Dimethyl iodovinyl active ester 7b was found to be exclusively a single isomer. A coupling constant of 15 Hz for the two vinyl protons indicated trans double bond geometry. Radioiodination of the vinyltin active esters 6a and 6b was accomplished in methanol solution using N-chlorosuccinimide (NCS) and nca (no carrier added) NalZ5I (or Na1311) to afford the desired labeled products 7a and 7b in yields ranging from 70% to 95%. Coinjection of the radiolabeled compounds and the corresponding standards on a reversed-phase HPLC system monitored by in-line UV and gamma det,ectors established the identity of the radiolabeled compounds. Also, HPLC analysis revealed that labeled 7a was a 45/55 mixture of isomers, while labeled 7b was a single isomer. The addition of 5% acetic acid to the methanol solution stabilized the reactive TFP esters toward hydrolysis and transesterification. Further, the addition of PBS to the reaction medium appeared to improve the reaction kinetics and the radiochemical yields. The reactions were quenched by the addition of a stoichiometric amount of NaHSO, based on the amount of NCS used. Quenching ensured that antibody was not exposed to electrophilic iodine species or excess oxidant during the conjugation reaction. Reaction of either iodovinyl reagent 7a or 7b with several antibodies or their Fab fragments afforded corresponding radiolabeled conjugates in yields ranging from 25% to 50%. Several different buffers, pH values, and reaction temperatures were evaluated for the conjugation reaction. Optimum conjugation yields were obtained by incubation of 7a or 7b with the desired antibody in pH 9.2 sodium carbonate buffer at 37 "C for 30 min. The reaction was quenched by the addition of a glycine solu-



Table I. Tissue Distribution of Radioiodine at 4 and 20 h Postcoinjection of Iodovinyl NR-ML-05 Fab Conjugate, [12'I]-7a, and PIB NR-ML-05 Fab Conjugate, [l3lI]-Zn' 20 h

4h

tissue blood tail tumor skin muscle lung liver spleen stomach neck kidney intestine

[lZ6I]-7a 1.54 f 0.16 2.08 f 0.31 4.79 f 0.91 1.66 f 0.29 0.69 f 0.18 1.94 f 0.31 0.59 f 0.11 0.73 f 0.06 2.03 f 0.21 1.90 f 0.28 6.32 f 0.93 0.50 f 0.06

[13111-2 1.27 f 0.15 1.93 f 0.33 4.49 f 0.90 1.28 f 0.13 0.53 f 0.11 1.90 f 0.39 0.51 f 0.09 0.44 f 0.05 0.48 f 0.18 0.80 f 0.10 11.99 f 2.00 0.91 f 0.29

[1z61]-7a 0.14 f 0.04 0.16 f 0.03 2.44 f 0.67 0.08 f 0.03 0.03 f 0.00 0.32 f 0.13 0.07 f 0.01 0.12 f 0.03 0.17 f 0.08 1.30 f 0.58 0.16 f 0.03 0.03 f 0.01

Table 11. Tissue Distribution of Radioiodine at 4 and 20 h Postcoinjection of Iodovinyl NR-ML-05Fab Conjugate, [ 1J11]-7a,and ['2~I]Iodide/Chloramine-T-Labeled NR-ML-05 Fabe-"

Results were obtained from n = 4 mice per time point and are tabulated as mean f SD of the percent injected dose/g. The specific activity and radiochemical purity for [1261]-7a and [1311]-2conjugates were 0.73 pCi/pg, 98.7%, and 0.94 pCi/pg, 99.5%, respectively. Tumor weights were 0.126 f 0.039 g.

tion studies were performed in athymic nude mice bearing human A375 Met Mix tumor xenografts. The first study compared the biodistribution of NRML-05 Fab labeled with the simple iodovinyl reagent 7a and NR-ML-05 Fab labeled with the PIB reagent (2). To make direct comparisons, the antibody was labeled with [lZ5I]-7aor [1311]-2, and the two preparations were subsequently combined and coinjected into the mice such that each animal received a 5-pg dose of each labeled preparation. Sacrifices of four animals were made a t 4 and 20 h postinjection. The data obtained from the biodistribution study are presented in Table I. Equivalent tumor uptake was evident for both labels a t both time points. However, significant differences in the biodistribution of the two labels were seen in the stomach, neck (thyroid),and kidney tissues. In the stomach tissue there was approximately a &fold greater accumulation of the iodovinyl label compared to the PIB label a t both time points. Also, in the neck tissue there was a 2-fold greater accumulation of the iodovinyl label compared to that of the PIB label a t the 4 h time point, and by 20 h, this differential increased to 14-fold. The 2-fold greater kidney uptake seen for the PIB label compared to that of the iodovinyl label a t the 4 h time point was noteworthy. A second study compared the biodistribution of NRML-05 Fab labeled with simple iodovinyl reagent 7a and NR-ML-05 Fab directly labeled by using the chloramineT method. As in the previous study, direct comparisons were performed by coinjection of the [1311]iodovinylconjugate of NR-ML-05 Fab and [1251]NaI/chloramine-T labeled NR-ML-05 Fab. The data obtained from this biodistribution study are presented in Table 11. The biodistributions of the two labels were remarkably similar in all tissues evaluated, including the tumor and kidney. Minor differences were seen in the biodistribution of the labels in the stomach and the neck tissues at both time points. Finally, a third study compared the biodistribution of NR-ML-05 Fab labeled with simple iodovinyl7a and NRML-05 Fab labeled with dimethyl iodovinyl reagent 7b. In this study, the mice were coinjected with the [ '311]iodovinyl conjugate of NR-ML-05 Fab and the dimethyl [ 1251]iodovinylconjugate of NR-ML-05 Fab. The results from this study are presented in Table 111. Equivalent tumor uptake was evident for both labels at both time points. However, there was approximately a 2-fold greater amount of iodovinyl label compared to the amount

20 h

4h

[1311]-2 0.13 f 0.03 0.14 f 0.05 2.19 f 0.67 0.04 f 0.00 0.01 f 0.00 0.28 f 0.12 0.03 f 0.00 0.01 f 0.01 0.03 f 0.01 0.09 f 0.03 0.24 f 0.03 0.04 f 0.01

159

tissue blood tail tumor skin muscle lung liver spleen stomach neck kidney intestine

113111-7a

112S11-Ch-T

1'3111-7a

2.86 f 0.24 2.79 f 0.33 4.40 f 0.37 1.87 f 0.35 0.91 f 0.07 2.73 f 1.19 0.85 f 0.05 1.08 f 0.07 8.39 f 1.65 3.80 0.44 6.16 f 1.01 0.77 f 0.10

2.55 f 0.23 2.82 f 0.33 4.27 f 0.42 1.86 f 0.25 1.02 f 0.28 2.84 f 0.88 0.84 f 0.06 1.20 f 0.10 10.21 f 2.46 4.25 f 0.58 5.68 f 1.03 0.81 f 0.12

0.11 f 0.01 0.13 f 0.05 1.38 f 0.31 0.07 f 0.01 0.02 f 0.00 0.21 f 0.06 0.05 f 0.00 0.07 f 0.04 0.17 f 0.07 1.73 f 0.29 0.11 f 0.01 0.03 f 0.01

*

[12611-Ch-T 0.09 f 0.01 0.14 f 0.05 1.27 f 0.27 0.06 f 0.01 0.02 f 0.01 0.32 f 0.13 0.04 f 0.00 0.06 f 0.05 0.18 f 0.07 2.12 f 0.39 0.07 f 0.00 0.03 f 0.01

a Results were obtained from n = 4 mice per time point and are tabulated as mean f SD of the percent injected dose/g. The specific activity and radiochemical purity for [I3lI]-7aand [12SI]iodide/ Ch-T conjugates were 0.76 pCi/pg, 98.0%, and 0.98 pCi/pg, 99.4%, respectively. Tumor weights were 0.074 f 0.017 g.

Table 111. Tissue Distribution of Radioiodine at 4 and 20 h Postcoinjection of Iodovinyl NR-ML-05 Fab Conjugate, [13'I]-7a, and Dimethyl Iodovinyl NR-ML-05 Fab Conjugate, [lzsI]-7b.-c

blood tail tumor skin muscle lung liver spleen stomach neck kidney intestine

3.42 f 0.69 2.79 f 1.01 4.85 f 0.34 1.80 f 0.36 1.09 f 0.09 1.77 f 0.34 0.84 f 0.15 1.17 f 0.19 9.13 f 0.29 3.21 f 0.95 5.60 f 0.13 0.61 f 0.10

2.85 f 0.41 2.61 f 1.00 5.00 f 0.35 1.57 f 0.18 0.94 f 0.12 1.74 f 0.33 0.91 f 0.12 1.14 f 0.09 4.81 f 0.24 2.07 f 0.46 6.34 f 0.31 0.55 f 0.08

0.12 f 0.02 0.16 f 0.08 1.53 f 0.56 0.07 f 0.01 0.03 f 0.01 0.25 f 0.18 0.05 f 0.02 0.05 f 0.02 0.33 f 0.17 3.17 f 1.32 0.14 f 0.04 0.04 f 0.01

0.10 f 0.02 0.19 f 0.11 1.79 f 0.55 0.05 f 0.01 0.02 f 0.01 0.34 f 0.31 0.09 f 0.01 0.13 f 0.04 0.19 f 0.10 1.58 f 0.58 0.11 f 0.04 0.03 f 0.01

a Results were obtained from n = 4 mice per time point and are tabulated as mean f SD of the percent injected dose/g. The specific activity and radiochemical purity for [1311]-7aand [12SI]-7b conjugates were 0.76 pCi/pg, 98.0%, and 0.19 pCi/pg, 99.0%, respectively. Tumor weights were 0.074 f 0.020 g.

of dimethyl iodovinyl label in the stomach a t 4 h and in the neck (thyroid) at 20 h. In all other tissues evaluated the biodistributions of the labels were essentially equivalent. DISCUSSION

Two iodovinyl protein radioiodination reagents 7a and 7b were prepared by radioiododestannylation of vinyltin active ester precursors 6a and 6b. The reactions are rapid and reproducibly afford high yields of the reagents 7a and 7b for either lZ5Ior 1311 labeling at the nca levels. Precursor vinyltin active esters 6a and 6b were synthesized in three steps from the corresponding pentynoic acids 3a and 3b in 26% and 47% overall yield, respectively. Reaction of 7a and 7b with NR-ML-05 Fab and other monoclonal antibodies or antibody fragments at basic pH afforded the desired antibody conjugates in yields ranging from 25% to 50%. The conjugation reaction was quenched by the addition of a glycine solution prior to gel filtration purification. The glycine quench ensured that the radiochemical purities of the purified antibody conjugates would consistently exceed 98% postpurification, as assessed by ITLC. The conjugation yields of 7a

160


and 7b were routinely lower than the conjugation yields of 2. These lower yields are probably due to a combination of the lower reactivity of a TFP active ester relative to an NHS active ester and to the more lipophilic, less soluble nature of 7a and 7b as compared to 2. We had previously found that the corresponding T F P ester of 2 was much less reactive and substantially more lipophilic than the NHS ester of 2 (24). The iodovinyl antibody conjugates were evaluated by several different in vitro procedures. The procedures included HPLC, SDS-PAGE combined with autoradiography, chemical challenge studies, serum stability studies, and immunoreactivity analysis. These studies demonstrated that the iodovinyl radiolabel was attached to the antibody in a stable manner. Also, the results demonstrated that the biological properties of the iodovinyl antibody conjugates, including immunoreactivity, were not compromised by the radiolabeling procedure. Biodistribution studies were performed to determine the in vivo stability and tumor localization of an antibody fragment labeled with iodovinyl reagents 7a and 7b and to compare their biodistributions to the antibody fragment labeled with PIB (2) or directly labeled by using the chloramine-T method. Dual-label studies were performed to minimize animal to animal variability within the studies. Radioiodide is known to accumulate in the thyroid and to a lesser extent in the stomach (25). Therefore, preferential accumulation of either radiolabel in these tissues would suggest greater in vivo deiodination. The results suggested that there was a greater amount of deiodination of NR-ML-05 Fab labeled with simple iodovinyl reagent 7a, as compared to NR-ML-05 Fab labeled with 2 (see Table I). The study suggested negligible in vivo deiodination of NR-ML-05 Fab labeled with 2, consistent with our previous findings (8). The amount of deiodination of NR-ML-05 Fab directly labeled by using the chloramine-T method and NR-ML-05 Fab labeled with the simple iodovinyl reagent 7a was almost equivalent (see Table 11). This result is somewhat surprising, since one might anticipate that quite different metabolic pathways would be responsible for the loss of label in each case (26). The data suggests that there was approximately a 50% reduction in the amount of deiodination of NR-ML-05 Fab labeled with 7b, as compared to that of NR-ML-05 Fab labeled with 7a. Although not directly measured, the amount of in vivo deiodination of NR-ML-05 Fab labeled with 7b appears to be approximately twice that of NR-ML-05 Fab labeled with 2. Finally, the results of all the studies demonstrated that, regardless of the labeling method employed, essentially equivalent tumor localization was seen. The stability of iodovinyl moieties has been demonstrated in vivo for the iodovinyl estradiols, where at 24 h postinjection in rats only 0.37% of the injected dose was found in the thyroid (12). Further, it has been reported that significant in vivo deiodination did not occur with iodovinyl fatty acids until 2 h after injection (11). It is not apparent whether it is the radioiodinated Fab conjugates of 7a and 7b or their lower molecular weight metabolites which are deiodinated in vivo. Deiodination of Fab labeled with 7b was demonstrably less than that of Fab labeled with 7a. Fab labeled with 7b, the P,p-dimethylpentenoic acid derivative, might have more effectively resisted deiodination if metabolism via enzymatic p-oxidation occurred, as previously shown for branched fatty acids (27). The preferential kidney uptake and/or retention of the PIB (2) label as compared to that of the simple iodovi-

Hadley and Wilbur

nyl (7a) label seen in the first biodistribution study is interesting. Similar differences in kidney localization were found when another antibody, NR-LU-10 Fab, was labeled with 2 and compared to NR-LU-10 Fab labeled with either ATE or chloramine-T.l Antibody Fab fragments rapidly accumulate in the kidneys and are presumably metabolized in the same manner as other circulating proteins by renal cells. Renal cell metabolism of proteins results in complete degradation of the protein to the amino acid level (28). Since it is likely that renal cell uptake and protein metabolism of labeled NR-ML-05 Fab are not influenced by the labeling method, then a plausible explanation for the differential kidney uptake seen with the PIB label is that the PIB (2), iodovinyl (7a), dimethyl iodovinyl (7b), and chloramine-T metabolites of labeled NR-ML-05 Fab are released from, or are subsequently reabsorbed by, the renal cells at different rates. In summary, Fab conjugates of the iodovinyl reagents 7a and 7b were immunocompetent and successfully targeted tumor in vivo. The Fab conjugate of dimethyl iodovinyl reagent 7b was substantially more resistant to in vivo deiodination than directly labeled Fab or the Fab conjugate of the iodovinyl reagent 7a. However, the Fab conjugate of the PIB reagent (2) was clearly more resistant to in vivo deiodination than the Fab conjugate of dimethyl iodovinyl reagent 7b. It is important to recognize that these in vivo results were obtained with a Fab fragment and that studies with intact antibody or F(ab’), fragments may afford different results. However, one would predict that the relative propensity of each labeled conjugate to suffer in vivo deiodination, as observed in these Fab studies, would be similar for intact antibody or F(ab’), studies. However, the magnitude of the observed differences may vary. In addition, it should be noted that biodistribution studies performed with other antibodies and tumor models may afford different results, particularly in cases where antibody conjugates are actively internalized and metabolized by the tumor target. ACKNOWLEDGMENT

We would like to thank Don Axworthy, Denise DuPont, Leah Grant, Dr. Mary Ann Gray, Cathy Jackson, Karen Poole, Joan Schroeder, and Carmen Soikowski for their efforts in obtaining the biodistribution, stability, and SDSPAGE data presented herein. We would like to also thank Dr. Alan R. Fritzberg, Dr. A. Charles Morgan, Dr. Paul L. Beaumier, and Dr. Debra K. Leith for their discussions on the experiments and manuscript. LITERATURE CITED (1) Lashford, L. S., Davies, A. G., Richardson, R. B., Bourne,

S. P., Bullimore, J. A,, Eckert, H., Kemshead, J. J., and Coakham, H. B. (1988) A Pilot Study of 13’1 Monoclonal Antibodies in the Therapy of Leptomeningeal Tumors. Cancer 61, 857-868. (2) Epenetos, A. A., Munro, A. T., Stewart, S., Rampling, R., Lambert, H. E., McKenzie, C. G., Soutter, P., Rahemtulla, A,, Hooker, G., Sivolapenko, G. B., Snook, D., CourtenayLuck, N., Dhorkia, B., Krauz, T., Taylor-Papadimitriou, J., Durbin, H., and Bodmer, W. F. (1987) Antibody Guided Irradiation of Advanced Ovarian Cancer with Intraperitoneally Administered Radiolabeled Monoclonal Antibodies. J. Clin. Oncol. 5 , 1890-1899. (3) Colcher, D., Carrasquillo, J. A., Esteban, J. M., Sugarbaker, P., Reynolds, J. C., Siler, K., Bryant, G., Larson, s. M., and Scholm, J. (1987) Radiolabeled Monoclonal Antibody B72.3 Localization in Metastatic Lesions of Colorectal Wilbur, D. S., Hadley, S. W., Grant, L. M., and Hylarides, M. D., unpublished results.


Cancer Patients. I n t . J. Radiat. Appl. Instrum. [ B ]14,251252. (4) Carrasquillo, J. A., Krohn, K. A., and Beaumier, P. B., McGuffin, R. W., Brown, J. P., Hellstrom, I., and Larson, S. M. (1984) Diagnosis of and Therapy for Solid Tumors with Radiolabeled Antibodies and Immune Fragments. Cancer Treat. Rep. 68, 317-328. (5) Regoeczi, E. (1984) Methods of Protein Iodination. In Iodine Labeled Plasma Proteins Vol. I, pp 35-102, CRC Press Inc., Boca Raton, FL. (6) Krohn, K. A., Knight, L. C., Harwig, J. F., and Welch, M. J. (1977) Differences in the Site of Iodination of Proteins Following Four Methods of Radioiodination. Biochim. Biophys. Acta 490,497-505. (7) Engler, D., and Burger, A. G. (1984) The Deiodination of the Iodothryonines and of Their Derivatives in Man. Endocr. Reu. 5, 151-184. (8)Wilbur, D. S., Hadley, S. W., Hylarides, M. D., Abrams, P. G., Beaumier, P. A,, Morgan, A. C., Reno, J. M., and Fritzberg, A. R. (1989) Development of a Stable Radioiodinating Reagent to Label Monoclonal Antibodies for Radiotherapy of Cancer. J. Nucl. Med. 30, 216-226. (9) Zalutsky, M. R., and Narula, A. S. (1987) A Method for the Radiohalogenation of Proteins Resulting in Decreased Thyroid Uptake of Radioiodine. I n t . J. Radiat. Appl. Instrum. .. [ A ]381 1051-1055. (10) Coenen, H. H., Moerlein, S. M., and Stocklin, G. (1983) No-Carrier-Added Radiohalogenation Methods with Heavy Halogens. Radiochim. Acta 34, 47-68. (11) Knapp, F. F., Jr., Goodman, M. M., Kabalka, G. W., and Sastry, K. A. R. (1984) Synthesis and Evaluation of Radioiodinated (E)-18-Iodo-l7-octadecenoic Acid as a Model Iodoalkenyl Fatty Acid for Myocardial Imaging. J. Med. Chem. 27, 94-97. (12) Hansen, R. N., Seitz, D. E., and Botarro, J. C. (1982) E17-alpha-[1251]iodovinylestradiol:An Estrogen-Receptor Seeking Radiopharmaceutical. J. Nucl. Med. 23, 431-436. (13) Goodman, M. M., Callahan, A. P., and Knapp, F. F. Jr. (1987) Design, Synthesis and Evaluation of 2-Deoxy-2iodovinyl Branched Carbohydrates as Potential Brain Imaging Agents. J. Labeled Compd. 23, 243-245. (14) Black, T. H. (1983) The Preparation and Reactions of Diazomethane. Aldrichimica Acta 16, 3-10. (15) Cordes, C., Prelog, V., Troxler, E., and Westen, H. H. (1968) 190. Zur Kenntnis des Kohlenstroffringes; 1,1,6,6-Tetramethyl-cyclodecan und seine Derivate. Helu. Chim. Acta 51, 1663-1678. (16) Nozaki, K., Oshima, K., and Utimoto, K. (1987) Et,B-Induced Radical Addition of R,SnH to Acetylenes and Its Application to Cyclization Reaction. J. A m . Chem. SOC.109,25472549.


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(17) Lindmo, T., Boven, E., Cuttitta, F., Fedorko, J., and Bunn, P. A. Jr. (1984) Determination of the Immunoreactive Fraction of Radiolabeled Monoclonal Antibodies by Linear Extrapolation to Infinite Antigen Excess. J.Immunol. Method. 72, 77-89. (18) Beaumier, P. L., Neuzil, D., Yang, H. M., Noll, E. A., Kishore, R., Eary, J. F., Krohn, K. A., Nelp, W. B., Hellstrom, K. E., and Hellstrom, I. (1986) Immunoreactivity Assay for Labeled Anti-melanoma Monoclonal Antibodies. J. Nucl. Med. 27, 824-828. (19) Kabalka, G. W., Sastry, K. A. R., Gooch, E. E. (1982) Synthesis of Very High SpecificActivity Radioiodinated and Radie brominated Materials via Organoborane Chemistry. J. Labelled Compd. 19, 1506. (20) Knapp, F. F., Jr., Goodman, M. M., Callahan, A. P., Ferren, L. A., Kabalka, G. W., and Sastry, K. A. R. (1983) New Myocardial Imaging Agents; Stabilization of Radioiodine as a Terminal Vinyl Iodide Moiety on Tellurium Fatty Acids. J . Med. Chem. 26, 1293-1300. (21) Charleson, F. P., Flanagan, R. J., Synnes, E. I., and Weibe, L. I. (1984) Rapid No Carrier Added Radiolabeling Using Organomercury Compounds. J. Labelled Compd. 21, 10741075. (22) Flanagan, R. J., Charleson, F. P., Synnes, E. I., and Weibe, L. I. (1986) Radiolabeling with Organomercury Compounds: Part 2. High Specific Activity Synthesis of Iodine-125 and Iodine-131-6-iodocholest-5-en-3-beta-ol and its Tissue Distribution in Rats. J . Nucl. Med. 27, 1165-1171. (23) Corey, E. J., Ulrich, P., and Fitzpatrick, J. M. (1976) A Stereoselective Synthesis of (rt)-ll-Hydroxy-trans-8decanoic Acid Lactone, a Naturally Occurring Macrolide from Cephalosporium recifei. J. A m . Chem. SOC.98, 222-224. (24) Hadley, S. W., Grant, L. M., and Wilbur, D. S. (1987) Evaluation of Radioiodinations and Conjugations of 4-Iodobenzoates for Protein Labeling. J. Nucl. Med. 28, 725. (25) Zalutsky, M. R., and Narula, A. S. (1988) Radiohalogenation of a Monoclonal Antibody Using an N-Succinimidyl 3(Tri-n-butylstannyl)benzoate Intermediate. Cancer Res. 48, 1446-1450. (26) Schulz, H. (1983) Metabolism of 4-Pentenoic Acid and Inhibition of Thiolase by Metabolites of 4-Pentenoic Acid. Biochemistry 22, 1827-1832. (27) Otto, C. A., Brown, L. E., and Scott, A. M. (1985) Radioiodinated Branched-Chain Fatty Acids: Substrates for Beta Oxidation? Concise Communication. J. Nucl. Med. 25, 7580. (28) Maack, T., Park, C. H., and Camargo, M. J. F. (1985) Renal Filtration, Transport, and Metabolism of Proteins. T h e Kidney: Physiology and Pathophysiology (D. W. Sheldin, and G. Giebisch, Eds.) pp 1773-1802, Raven Press, New York.

Evaluation of iodovinyl antibody conjugates - American Chemical

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