Bioconjugate Chem. 1993, 4, 275-283
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Synthesis, Metal Chelate Stability Studies, and Enzyme Digestion of a Peptide-Linked DOTA Derivative and Its Corresponding Radiolabeled Immunoconjugates Min Li and Claude F. Meares* Department of Chemistry, University of California, Davis, California 95616-0935. Received March 22, 1993
By directly coupling a tetrapeptide to DOTA through an amide bond, we synthesized a novel DOTA derivative, DOTA-glycylglycylglycyl-L-p-nitrophenylalanine amide. We converted this new precursor and conjugated bifunctional chelating agent to DOTA-glycylglycylglycyl-L-p-isothiocyanatophenylalanine it to monoclonal antibody Lym-1. Serum stability studies show that the radiolabeled conjugates are kinetically inert under physiological conditions. The rates of loss of radiometals in human serum are 0.1 f 0.1% per day for In”’, 0.02 f 0.15% per day for YIII, and 0.3 f 0.2% per day for Cu” labeled immunoconjugates. In the presence of the liver enzyme cathepsin B, an in vitro digestion of 114mInlabeled conjugate yields a small fragment containing 114mIn.Characterization of the cleavage products shows that this liver enzyme hydrolyzes the peptide linkage before the phenylalanine residue, freeing the In-DOTA-triglycine complex from the conjugate. However, the liver enzyme cathepsin D does not cleave the linkage over the span of 7 days.
INTRODUCTION Bifunctional chelating agents (BCAsl) which contain a chemicallyreactive functional group for binding to proteins and a strong metal chelating group have been used to label monoclonalantibodies (mAbs)with radiometals for cancer diagnosis and therapy (1-5). Metallic radioisotopes such as “‘In, ~ T c“Cu, , my, and 6sGa are primary candidates for 1abelingmAbsdue to their favorable physical properties (4,6). BCAs in which the chelating moiety can hold radiometals with high stability under physiological conditions are essential to avoid excessive radiation damage to nontarget cells (7). In recent years, macrocyclic poly(amino carboxylate) BCAs have gained considerable attention (8-13).One of the most promisingagents studied so far is DOTA; its derivatives form extraordinarily stable complexeswith a variety of metal ions under physiological conditions (9, 12, 14). Several DOTA derivatives have been synthesized, each synthesis involving multiple steps (9, 10, 15, 16). Another major detriment to the use of radiometallabeled mAbs for radiopharmaceutical applications is the high accumulation of radioactivity in the normal organs, particularly the liver (17-20).One approach to reducing the liver uptake of radioactivity is to attach the chelate to the antibody through a readily metabolizable chemical linker (21-23). This may accelerate the cleavage of the chelate from the antibody when the antibody-chelate conjugate is endocytosed into an enzyme-rich intracellular compartment (21,24).Once cleaved, the chelated radiometal may be returned to the circulation and rapidly excreted through the kidney. This paper describes the chemical and biochemical properties of a novel DOTA derivative containing a cleavable linker. This new BCA does not contain a carbon1 Abbreviations used: BAT, 6-@-(bromoacetamido)benzyl)1,4,8,11-tetraazacyclotetradecane-ZV,”,”,N”-tetraacetic acid; BCA, bifunctional chelating agent; DOTA, 1,4,7,1O-tetraazacyclododecane-Nfl,”,N”-tetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; EDTA, ethylenediaminetetraacetic acid; 2IT, 24minothiolane; PBS, phosphate-buffered saline. TMSP, 3-(trimethysilyl)propionic-2,2,3,3,-d4 acid, sodium salt.
1043-1802/93/2904-0275804.o010
linked side chain; the linker is attached through an amide bond to a DOTA carboxyl group (see Scheme I). EXPERIMENTAL PROCEDURES Chemicals. Triglycine, cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5), and dithiothreitol were purchased from Sigma Chemical Co. 2- [[(tert-Butoxycarbonyl)oxyliminol-2-phenylacetonitrile,p-nitrophenol, isobutyl chloroformate, palladium on charcoal (10%1, thiophosgene, and DTPA were obtained from Aldrich. DOTA (sodium salt) was bought from Parish Chemical Co. and its purity (87.2%) was determined by a Cobalt-57 binding assay (25). Disodium EDTA and CFBCOOHwere obtained from Fisher Scientific. DTPA anhydride was purchased from Pierce. Peptide substrate for cathepsin D, BOC-Phe-Ala-Ala-(p-NO2)Phe-Phe-Val-Leu-4-(hydroxymethy1)pyridine was bought from Research Plus, Inc. Lym-1, an anti B cell lymphoma IgGzamAb (26),was obtained from Damon Biotech (Needham Heights, MA; Encapcel murine mAb, lot #3-171-860813). Lym-1 was further purified by protein A affinity column chromatography prior to use. 57C~C12, WuC12, l14”InC13, and 88YC13were purchased from ICN, Brookhaven, Du Pont, and Los Alamos, respectively. Pure water (18 Mil cm-l) was used throughout. All glass labware was washed with a mixed acid solution and thoroughly rinsed with deionized, distilled water (27). All plastic labware was washed with 3 M HC1 and thoroughly rinsed. All other chemicals were the purest grade available. Thin-Layer Chromatography. TLC was run on plastic-backed silica gel plates (Kieselgel 60 F ~ MEM , Science) using various solvents as described below. For the metal-binding assays, a solution containing equal volumes of 10% (w/v) aqueous ammonium acetate and methanol was used as the eluent. High-Performance Liquid Chromatography. HPLC was performed at room temperature on a Rainin HPXL system or a Waters HPLC instrument (pump model M-45, controller model 660). The columns,gradients,and solvent systems are described below. The UV-absorbingfractions were detected either at 254 nm or a t 280 nm using an ISCO UA-5 detector. 0 1993 Amerlcan Chemical Society
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Bloconjugate Chem., Vol. 4, No. 4, 1993
Scheme I I-BOCQN c
HaN
NH
1-BOGHN
NH
NH
OH
6
CSCI,. 85% In CCl, 3M HCI
NCS
7
Radiation Counting. y-counting was done with a Beckman Model 310 counter. TLC plates and protein gels containing radiolabeled materials were visualized with an AMBIS Radioanalytical Imaging System. The HPLC effluent was monitored by a Beckman 170 flow-through radioisotope detector. Spectroscopy. NMR spectra were recorded on a General Electric QE 300 spectrometer at 300 MHz for ‘H
and at 75 MHz for ‘3C. The chemical shifts are relative to the monoprotonated solvents (HOD,4.80ppm; CDsOH, 3.30 ppm; CDs(SO)CD2H, 2.49 ppm) and TMSP as an external reference. Reported pH meter readings are not corrected for the deuterium isotope effect. Mass spectra and exact mass measurements were obtained on a ZAB-HS-2F mass spectrometer (VG Analytical, Wythenshawe, UK). During mass spectroscopic measurements, either 3-nitrobenzyl alcohol or dithiothreitol/dithioerythritol(3:1w/w) was used as a matrix along with small amounts of p-toluenesulfonic acid. IR spectra were performed on an IBM 32 FTIR spectrophotometer. Concentrations of antibody (MW 155 kDa) were determined by absorbance a t 280 nm (E1%2w = 13.5 (28)). The measurements were recorded on a Gilford Model 250 spectrophotometer using a 1-cm pathlength microcell. The cathepsin D enzyme activity assay was performed on an H P 8450A UV/vis spectrophotometer. Bifunctional Chelating Agent Synthesis. N-(tertButoxycarbony1)glycylglycylglycine( 1). Triglycine (29.25 g, 154.8 mmol) and triethylamine (32.3 mL, 154.8 X 1.5 mmol) in 100 mL of water was mixed with 2-[[(tertbutoxycarbonyl)oxylimino]-2-phenylacetonitrile (42.47 g, 154.8 X 1.1 mmol) in 100 mL of dioxane a t room temperature under N2. After stirring for 6 h, 100 mL of water and 100 mL of ethyl acetate were added. The aqueous phase was washed with ethyl acetate ( 5 X 200 mL) and then acidified to pH 1 using 6 M HC1 and extracted with ethyl acetate (10 X 200 mL). The extracts were taken to dryness under reduced pressure. The residue was washed with ethyl ether and allowed to air dry at room temperature to yield 1 as a white solid: yield, 32.8 g, 73%; ‘H NMR (D2O) 6 1.35 (8, 9H), 3.75 (8, 2H), 3.95 (s,4H); FAB-MS m/e = 290 (M + H+). N-(tert-Butoxycarbony1)glycylglycylglycinep-Nitrophenyl Ester (2). p-Nitrophenol(O.865 g, 5.51 X 1.2 mmol) was added to a solution of 1 (1.50 g, 5.15 mmol) in 25 mL of pyridine. Then 1,3-dicyclohexylcarbodiimide (1.4 g, 5.15 X 1.2 mmol) was added at 0 OC under N2. The reaction temperature was kept at 0 “C for 30 min, then at room temperature for 4.5 h. The precipitate was filtered off and the filtrate was evaporated to dryness under reduced pressure. The residue was taken up in hot 2-propanol and upon cooling, 2 crystallized as a white solid: yield, 1.68 g, 79%; TLC (eluent, CHClg/ethyl acetate/CHsOH 5 5 1 v/v/v) Rf = 0.24; lH NMR (CDsOD) 6 1.35 (8, 9H), 3.75 (8, 2H), 3.95 (8, 2H), 4.25 (8, 2H), 7.45 (d, 2H), 8.30 (d, 2H); FAB-MS m/e = 411 (M + H+). N-(tert-Butoxycarbony1)glycylglycylglycy1-L-pnitrophenylalanine Amide (3). L-p-Nitrophenylalanine amide (112 mg, 0.488 X 1.1mmol), prepared according to Yeh et al. (291,was added to a solution of 2 (200 mg, 0.488 mmol) in 5 mL of NJV-dimethylformamide a t room temperature. After stirring for 5 h, the solvent was evaporated to dryness under reduced pressure and the resulting yellow residue was washed with CHsOH five times to give 3 as a white precipitate: yield, 120 mg, 51 5% ;TLC (eluent, CHC13/CH30H 1:l v/v) Rf = 0.60; ‘H NMR (dimethyl sulfoxide-dslDaO 1:l) 6 1.35 (8, 9H), 2.90 (m, lH), 3.15 (m, lH), 3.6-3.8 (m, 6H), 4.40 (m, lH), 7.45 (d, 2H), 8.10 (d, 2H); FAB-MS mle = 481 (M + H+). Glycylglycylglycyl-L-pnitrophenylalanine amideTrifluoroacetic Acid (4). Compound 3 (94 mg, 0.196 mmol) was dissolved in 5 mL of neat CFsCOOH at 0 “C and the solvent was removed after 1 h under reduced pressure. The residue was lyophilized twice from water to give 4 as a white solid in quantitative yield: lH NMR
Bioconjupte Chem., Vol. 4, No. 4, 1993 277
PeptMe-Linked DOTA
Table 1. Radiolabeling Immunoconiugates starting conjugate specific chelates radioactivity, labeling labeling challenge chelate final conc, activity, conjugate per mAb radioisotope pCi buffer conditions stock solution mg/mL mCi/mg 114mIn 26 0.1 M NHAOAC. 2 h RT 100 mM EDTA in 0.94 0.018 pH = 5:5 ' 2 M (NH4)scitrate DOTA-G~~~-LMY 24 2.0 M NH~OAC, 3 h RT 50 mM DTPA in 0.94 0.026 Phe-thiourea- 1.2 f 0.1 pH = 7.0 0.2 M (NH&citrate Lym-1 6'CU 250 0.1 M (NH&citrate, 0.5 h RT 100 mM EDTA in 0.73 0.65 pH = 5.0 2 M (NH&itrtae ~
~~
33
DTPA-Lym-1
3.5 f 0.1
sy
0.5 M NH~OAC, pH = 5.5
26
BAT-2IT-Lp-1
2.2 f 0.1
"CU
131
114mIn
1 h RT
0.63
0.14
0.1 M (NHd)scitrate, 1 h RT pH = 6.5
100 mM EDTA in 2 M (NH&citrate 50 mM DTPA in 0.2 M (NH4)scitrate
0.39
0.031
0.1 M (NH&itrate, pH = 5.0
100 mM EDTA in 2 M (NH4)scitrate
0.23
0.61
0.5 h RT
100% solvent B (solvent A, 0.1% CF3COOH in H2O; (D20) 6 3.15 (m, lH), 3.35 (m, lH), 3.8-4.1 (m, 6H), 4.65 (m, lH), 7.45 (d, 2H), 8.20 (d, 2H); FAB-MS m / e = 381 solvent B, 0.1% CF3COOH in CHsOH), was used. Two C O O peaks H - were collected, at retention times 25.0 and (M H+). Anal. Calcd for C ~ ~ H ~ O N ~ O ~ ' ~ / ~ C F ~ major 27.0 min. The second major peak at 27.0 min was dried H2O: C,38.55;H,4.27;N, 15.27;F, 13.81. Found:C,38.63; to give 7 as a white solid: yield, 8.6 mg, 175% by 57Co binding H, 4.28; N, 15.23; F, 14.04. assay; 'H NMR (D20, pH 1.4) 6 2.8-4.3 (m, 32H), 4.60 (m, DOTA-Glycylglycylglycyl-L-p-nitrophenylalalH), 7.20 (d, 4H); FAB-MS m / e for C32H~N9012S(M + nine Amide (5). DOTA sodium salt (81 mg, 0.20 mmol) H+) = 780.2987, found 780.2997; IR (KBr) 2118 cm-1 was dissolved in 10 mL of aNa-dimethylformamide and ( v N ~ + , ) . Anal. Calcd for C32H4sNg012S.1.42CF3COOH: triethylamine mixture (4:l v/v) overnight at room temC, 44.44; H, 4.97; N, 13.39; F, 8.58; S, 3.41. Found:C, perature. Isobutyl chloroformate (26.5 pL, 0.20 mmol) 44.73; H, 4.90; N, 13.72; F, 9.01; S, 3.60. The first peak was was added dropwise at 0 "C to the mixture. After 15 min, dried to give DOTA-glycylglycylglycyl-L-p-isothiocy4 (98 mg, 0.20 mmol) was added and the reaction was kept anatophenylalanine amide (8) as a white solid: yield, 19.8 at room temperature for 5 h. The reaction mixture was mg, 39% by W o binding assay; lH NMR (D20, pH 1.4) purified by reversed-phase HPLC using a 21.4 X 250 mm 6 2.8-4.3 (m, 32H), 4.50 (m, lH), 7.20 (d, 4H); FAB-MS c18 column (Dynamax 60 A) with a flow rate of 12.5 mL/ min. A 20-min linear gradient, from 15% to 100% solvent m / e for C32H47N10011S (M + H+) = 779.3147, found B (solvent A, 0.1 M aqueous ammonium acetate, pH 6.0; 779.3150; IR (KBr) 2116 cm-' (vN-c-s). Calcd for solvent B, CH30H) was used. The major peak at retention C ~ ~ H ~ ~ N ~ O O ~ ~ S . ~ /C, ~ C44.73; F ~ CH,O5.13; O H N, : 15.05; time 10.5 min was collected and dried to give 5 as a light F,8.16;S,3.44. Found: C,45.00;H,5.22;N, 14.93;F,9.88; S, 3.05. Both products were reinjected on an HPLC using yellow solid: yield, 68 mg, 45 % by weight; purity, 96.2 % by WObinding assay (25);'H NMR (D20, pH 6.0) 6 3.6a 10 X 250 mm C18 column (Dynamax 60 A) with a flow 4.1 (m, 32H), 4.65 (m, lH), 7.45 (d, 2H), 8.20 (d, 2H); 13C rate of 3.0 mL/min. A 30-min linear gradient, from 15% to 100% solvent B (solvent A, 0.1% CF3COOH in H2O; NMR (D20, pH 6.0) 6 37.52, 43.42, 43.58, 49.18, 49.51, solvent B, 0.1 % CF&OOH in CH30H), was used. A single 51.65, 57.72, 54.88, 55.41, 57.13, 57.56, 124.56, 131.10, peak was observed for both cases. The retention times for 146.06,147.51,170.45,172.10,173.05,173.29,173.60,175.78, 176.82; FAB-MS m / e for C31H47N10013 (M + H+) = product 7 and 8 were 24 and 23 min. Preparation of DOTA-Gly-Gly-Gly-L-Phe-Thio767.3324, found 767.3352. Anal. Calcd for C31HaN13013: C, 48.56; H, 6.05; N, 18.27. Found: C, 48.64; H, 6.16; N, urea-Lym-1 conjugate (9). An aqueous solution of 7 (50pL, 15.14 mM) was added to 1.5 mL of Lym-1 antibody 18.15. solution (16 mg/mL) in 0.1 M tetramethylammonium DOTA-Glycy lglycylglycyl-L-paminop henylalaphosphate buffer, pH 8.0. The Lym-1 antibody solution nine Amide (6). Compound 5 (50 mg, 65 pmol) was dissolved in 15 mL of water and the pH was adjusted to had been prepared by exchanging the phosphate-buffered saline with storage buffer using a Sephadex G-50centri11.5 with 0.2 M NaOH. The reaction was placed in an ice fuged gel-filtration column (25,30).After mixing of the bath and 1mg of 10% Pd/C was added to the cold solution. reactants, the pH was adjusted to 8.89 with 2 M triethThe mixture was stirred under 1atm of H2 for 8 h a t 0 "C. anolamine and the reaction mixture was incubated at 37 The reaction solution was neutralized with 1M HC1 and "C for 2.5 h. The conjugate 9 was then purified using then filtered through a 0.45-pm syringe filter (Nylon-66, Rainin) to remove the Pd/C catalyst. The filtrate was another centrifuged gel-filtration column. The degree of aggregation of the conjugate was determined by TSK 3000 lyophilized to give 6 as a white solid in quantitative yield: gel filtration HPLC (solvent, 0.1 M aqueous ammonium lH NMR (D20, pH 5.7) 6 2.7-4.1 (m, 32H), 4.50 (m, lH), acetate, pH 6.0; flow rate, 1.0 mL/min). The immuno6.90 (d, 2H), 7.10 (d, 2H); FAB-MS m / e = 737 (M + H+), conjugate peak (150 kDa) appeared at retention time 9.0 759 (M + Na+), and 781 (M + 2Na+). DOTA-Glycylglycylglycyl-L-pisothiocyanatophe- min and no aggregate was observed. Radiolabeling of DOTA-Gly-Gly-Gly-L-Phe-Thionylalanine (7) and DOTA-Glycylglycylglycyl-L-p isothiocyanatophenylalanineAmide (8). Compound urea-Lym-1. The radionuclides llenIn, =Y,andB7Cuwere used for labeling the conjugate. Labeling conditions are 6 (48 mg, 65 pmol) in 5 mL of 3 M HCl was added to 3 mL listed in Table I. Radiometal solutions (114mInand S7Cu of CSCl2 (855% in CC4) at room temperature. The reaction were in 0.5 M HC1, MY was in 6 M HC1) were dried under was stirred vigorously for 6 h. The aqueous phase was a heat lamp and dissolved in 2 pL of labeling buffer. To washed with chloroform (4 X 3mL) to remove excessCSC12 and then purified by reversed-phase HPLC using a 21.4 each of these solutions was added 18 pL of 9 (12.8 mg/mL) in the same labeling buffer and the mixture incubated a t X 250 mm CIScolumn (Dynamax 60 A) with a flow rate of 12.5 mL/min. A 40-min linear gradient, from 15% to room temperature for a certain period (see Table I). The
+
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Li and Meares
Bioconjugate Chem., Vol. 4, No. 4, 1993
solutions were then diluted with 115pL of labeling buffer, and 15 pL of challenge chelate stock solution was added to give a final concentration of 10 mM EDTA (or 5 mM DTPA). The solutions were incubated for another hour at room temperature, followed by purification and solvent exchange into H2O using a centrifuged gel-filtration column. An aliquot of each final solution was mixed with the same volume of challenge chelate solution. This mixture was incubated for 1 h and examined by TLC (eluent, 10% (w/v) NH40Ac/CH30H 1:l v/v; Rf = 0.0 for all the metal-conjugates,Rf = 0.70 for In-EDTA and CuEDTA, and R f = 0.60 for Y-DTPA ). The TLCs showed that the conjugate chelated each radioisotope quantitatively. Preparation and Radiolabeling of DTPA-Lym-1. The conjugate was prepared by the method of Hnatowich et al. (31). An aqueous solution of Lym-1 (24 mg) was dried under reduced pressure and then mixed with solid DTPA anhydride (1mg, 2.79 mmol). One mL of 0.1 M tetramethylammonium phosphate buffer, pH 7.0, was added. The reaction mixture was incubated at room temperature for 1 h. The DTPA-Lym-1 conjugate was then purified using a centrifuged gel-filtration column. The conditions for radiolabeling the DTPA-Lym-1 conjugate with lldmIn and 88Y are listed in Table I. was Radiometal solutions (114mInwas in 0.5 M HC1, in 6 M HC1) were dried under a heat lamp and dissolved in 5 pL of labeling buffer. To each of these solutions was added 15 pL of DTPA-Lym-1 (8.2 mg/mL) in the same labeling buffer and the mixture incubated for 1h at room temperature. The procedures for purification and characterization of radiolabeled conjugates were the same as described above for conjugate 9. Preparation and Radiolabeling of BAT-21T-Lym1. This conjugate was prepared according to the method of Deshpande et al. (32). The conditions for 67Culabeling are listed in Table I. The 67Cusolution (in 0.5 M HC1) was dried under a heat lamp and dissolved in 2 pL of labeling buffer. Then 4.6 pL of BAT-2IT-Lym-1(10.8 mg/mL) in the same labeling buffer was added and the mixture was incubated at room temperature for 0.5 h. The procedures for purification and characterization of 67Cu-BAT-21T-Lym-l were the same as described above for conjugate 9. Immunoreactivity Assay. Conjugate 9 was labeled with lllIn using the same labeling method as labeling with 114mIn. lllIn-labeled conjugate 9 and 67Cu-BAT-21TLym-1 were assayed for immunoreactivity by the solidphase radioimmunoassay method using '26I-labeled Lym-1 as a standard (33). The immunoreactivities of conjugate 9 and BAT-21T-Lym-1 were 89.9% and 85.5% relative to '25I-labeled antibody. Serum Stability Studies. Blood was collected from a healthy volunteer, allowed to clot for 1 h at room temperature in a closed tube, and then centrifuged. The supernatant serum was transferred to sterile plastic culture tubes and incubated at 37 "C in a humidified carbon dioxide incubator (Equatherm C02 incubator, Model 330). The pH of the serum was measured as 7.4 before aliquoting into separate sterile vials for the addition of radiolabeled Lym-1 conjugates. A 10-40-pL aliquot of radiolabeled conjugate was added to 1-2 mL of serum. The final concentrations of chelating agent are listed in Table 11. The samples were maintained at 37.0 f 0.2 "C and at pH 7.4 f 0.1 (air:COz was approximately 95:5 by volume)throughout the experiment. The stability of each conjugate in human serum was determined by analyzing small samples of serum at daily
Table 11. Results of Human Serum Stability Studies chelating agent finalconc, % conjugate nM loss/dap 123 0.05 In-DOTA-Gly3-L-Phe-thiourea-Lym-1 184 o.18 f 0.07 o.21 ~~~
In-DTPA-Lml
157
Y-DOTA-Glys-L-Phe-thiourea-Lym1
121
Y-DTPA-LD-1
Cu-DOTA-Glys-L-Phe-thiourea-Lym1 CU-BAT-2IT-Lym-1 a Errors are 95% confidence limits.
182 97 95 95 69
0.76 f 0.42 0.02 f 0.22 0.01 f 0.08 1.06 f 0.60 0.38f 0.19 0.19 f 0.14 0.85f 0.92
intervals for 2 weeks. Eight percent polyacrylamidenative protein slab gels (34) were used to resolve the different proteins in the serum samples. Bromophenol blue solution in adjacent lanes marked the dye front. The gels were dried, visualized on the AMBIS, cut into slices, and quantified using a y-counter. In VitroEnzyme Digestion of l1"1n-Labeled Lym-1 Conjugate 9 with Cathepsin B and Cathepsin D. Five microliters of 75 mM dithiothreitol and 12.5 pL of 0.3 M tetramethylammonium acetate, pH 4.8, were mixed with 2.5 pL of either cathepsin B (activity = 22 unita/mL (35)) or cathepsin D (activity = 2.2 units/mL (36)). After 15 min at room temperature, 30 pL of 114mInlabeled conjugate 9 in 0.1 M ammonium acetate, pH 4.8, was added to give a final concentration of 0.55 pM. The final activities of cathepsin B and cathepsin D were 1.1and 0.11 units/mL. After incubation at room temperature for 0.25, 1,12,18, 24,36,48,72,96, and 165 h, aliquots of the solutions were analyzed by TLC (eluent, 10% (w/v) NH40Ac/CH30H 2:l v/v). After 165 h of incubation, each digest solution was analyzed by reversed-phase HPLC to identify the cleav e products. A 4.6 X 250 mm Cla column (Dynamax 300 ) with a flow rate of 0.7 mL/min and a 30-min linear gradient from 5% to 100% solvent B (solvent A, 0.1 % CF3COOH in H2O; solvent B, 0.1 % CF3COOH in CH30H) was used. The cleaved products were monitored by both a UV detector and a flow-through radioisotope detector. Unsubstituted l14"In-DOTA was independently prepared and used as a standard. Cathepsin D Activity Assay. Because this enzyme showed no activity against the DOTA peptide linker, it was assayed to confirm that it was active against a standard substrate. The assay followed the method described by Agarwal et al. (37). Phe-Ala-Ala-@-NO2)Phe-Phe-ValLeu-44hydroxymethyl)pyridine was used as a peptide substrate. The rate of cleavage of the peptide was followed spectrophotometrically at 310 nm in 0.01 M sodium formate buffer, pH 3.5, at room temperature. The concentrations of the substrate and enzyme were 0.047 mM and approximately 0.005 pM. By comparison of the concentrations of enzyme found by weight and by assay, the enzyme activity assay shows that cathepsin D is 60% active.
x
RESULTS Synthesis. The synthesis of DOTA-glycylglycylglycylL-p-nitrophenylalanine amide (5) is shown in Scheme I. Because of its solubility in N&-dimethylformamide, L-pnitrophenylalanine amide was used as a starting material instead of L-p-nitrophenylalanine methyl ester. In the preparation of 5, a side product detected by HPLC, retention time 15 min, was also collected and identified
Bioconlugete Chem., Vol. 4, No. 4, I993
PeptMe-Linked DOTA
by mass spectroscopyas DOTA conjugated to two peptides. Treatment of 6 with CSClz not only converts the aromatic amine to the isothiocyanate but also partially hydrolyzes the primary amide. DOTA-glycylglycylglycyl-L-p-isothiocyanatophenylalanine (7) was found to have an HPLC retention time of 27.0 min (17% yield). Another major peak detected at 25.0 min was identified as DOTAglycylglycylglycyl-L-p-isothiocyanatophenylalanineamide (8) (39% yield). Conjugation and Radiolabeling. The ratios of chelate to Lym-1 for DOTA-Gly3-~-Phe-thiourea-Lym-l, DTPALym-1, and BAT-21T-Lym-1 are listed in Table I. The results were determined by UV absorbance and 57C0 binding assay (25). The final concentration of Lym-1 conjugates and the specific activity after radiolabeling are also listed in Table I. Serum Stability Studies. Serum mixtures of l14,1n-, MY-, and 67Cu-DOTA-Glya-~-Phe-thiourea-Lym-l at different times of incubation under physiological conditions were analyzed by electrophoresis. The relative mobility (R,) of the labeled antibody, transferrin, and albumin was found to be 0.22,0.40, and 0.60, respectively. The gel electrophoresis profiles showed that there were negligible counts in the R, 0.35-0.45 region throughout the experiment period for all three different radiometallabeled Lym-1peptide-DOTA conjugates. However, some counts were found in the R, 0.95-1.05 region, which were most likely due to free chelates cleaved from the antibody by hydrolysis. Deshpande et al. (14) reported a similar observation for Lym-2IT-2-(p-(bromoacetamido)benzy1)DOTA-MY. For comparison, 114mIn-DTPA-Lym-1, aY-DTPA-Lym- 1and Wu-BAT-21T-Lym-1 were prepared as described above and studied under the same conditions. The gel electrophoresis profiles for these radiolabeled conjugates showed that the counts in the transferrin R, 0.35-0.45 (for 114mIn and MY) or albumin R, 0.50-0.65 (for 67Cu)and Rm 0.9-1.1 regions increased to varying degrees with time. The sum of counts in R, 0-0.35 and 0.9-1.1 regions were divided by total counts to determine the percentage of intact metal chelates as a function of time. The results are plotted in Figure 1. Since the metals were present in trace amounts, the initial loss of metal from the chelate to serum proteins should follow pseudo-first-order kinetics (12). Table I1 lists the apparent rate constants corresponding to the curves shown in Figure 1. The plots in Figure 1 should be fit by exponential functions as exp(-kit), where ki is a pseudo-first-order rate constant and t is time. Since the rates of loss of metal from the chelates are quite slow, only a small fraction of the metal was lost during each experiment. Thus, the plots can be expressed 1 - kit]. by straight lines [when Kit 6) have been reported (451,including a coordination number of 8 in In-DTPA crystals (46). Since the crystal structure of InDOTA has not been determined, the fourth carboxylgroup may or may not coordinate to Inm. However, Riesen et 01. (45)have reported that the fourth carboxyl group of DOTA contributes little to the serum stability of In= DOTA. Therefore, chemical modifications on the fourth carboxyl group of InmDOTA may not change ita kinetic stability. YmDOTA may lose some degree of stability by this modification due to the high coordination number of Ym. By studying the related Gd(II1)-DOTA-propylamide complexes, Sherry et al. (47) have found that the conversion of a carboxylic group into an amide does not result in an increase in the number of inner-sphere water
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Bloconlugate Chem., Vol. 4, No. 4, 1993 281
designed to approximate the lysosomalenvironment found molecules. This suggests that the amide occupies a site in the human liver (pH 4.8-5.0, thiols present, and activities in the Gd(II1) coordination sphere and, although weakly of cathepsin B and cathepsin D 1.1-2.1 (58)and0.18 units/ bound, interacts well enough to preclude the entrance of mL (36)). additional water molecules into the inner coordination sphere. Aime et al. (48) also observed that the bond Figure 3 shows that cathepsin B efficiently cleaves the distances between carboxylate oxygen and carboxamide chelate from the protein, with an apparent half-life (T1,z) oxygen to Gd(II1) are quite similar on the basis of the of approximately 20 h. However, cathepsin D does not X-ray structure of a related Gd(II1)- DOTA-monoamide show any proteolytic activity to the conjugate over the complex. Becausethe properties of Ymare similar to those span of 7 days. From both TLC and HPLC profiles of lanthanide(II1) ions, it is reasonable to expect that YIn (Figures 2 and 4A) of the cathepsin B digestion, a single and Gd(II1) have similar strengths of interaction with cleaved product was observed. This cleaved product had the same HPLC retention time as the one cleaved from amide. Thus, the influence on the stability of YIILDOTA due to introduction of an amide is expected to be limited. 114mIn-DOTA-Gly3-~-@-NOz)Phe-NHz by cathepsin B (peak 1and peak 2 in Figure 4). This suggests that both The kinetic stability of metal chelates under physiocleavage products are identical and that the cleaved logical conditions can be estimated by study of their product from the conjugate does not contain any fragments behavior in serum. In this study, metal chelates at extreme of the protein. According to the mass spectrum, the dilution (1200 nM) are surrounding by metal-binding cleaved product is In-DOTA-glycylglycylglycine and the proteins, such as transferrin and albumin (49). If there site of cleavage is between glycine and phenylalanine. are any metals lost from the chelator, they are not likely Neither the di- nor the monoglycyl derivatives were to return (50,51).The gel electrophoresis profiles obtained observed. here show that the transcomplexation of 114mIn, or Since the simple linker Gly-Gly-Gly-Phe used in this B7Cu from the DOTA-peptide to transferrin or albumin study is not an optimal substrate for the cathepsins, future can nearly be ignored. The very low rate constants for loss of l14mIn-, MY-, and B7Cu-DOTA-Gly3-~-Phe-thio- studies will include both sequence-specific linkers that exhibit high cathepsin activity and in vivo biodistribution urea-Lym-1 listed in Table I1 suggest that amide-attached studies. The excellent metal-binding properties of amideDOTA derivatives are comparable to the C-functional or attached DOTA derivatives have shown that this new type N-functional derivatives (9,12,14)and therefore they are of BCA may offer practical advantages over other DOTA excellent prospects for in vivo use. derivatives. It also has been noted that carbon backbone-substituted Acknowledgment. We thank Douglas P. Greiner, EDTA or DTPA derivatives form In or Y-radiolabeled David L. Kukis, and Ani1 K. Mishra, for their technical complexesor immunoconjugates which have higher serum advice and helpful discussions, and Daniel Jones and Kei stability than the N-attached (including amide-attached) Miyano, for running the FAB-MS. This work was supderivatives such as the amides (9,52-54). These results ported by Research Grant CA16861 from the National may be explained by the increase in steric rigidity which Cancer Institute, NIH. helps to keep the metal in the chelator (55). However, this may be less important for DOTA derivatives since LITERATURE CITED DOTA itself possesses a sterically rigid structure (44,561. (1) Meares, C. F. (1986)Chelating Agents for the Binding of Using a cleavable linker between the chelating agent Metal Ions to Antibodies. Nucl. Med. Biol. 13,311-318. and the antibody is one approach to decrease the accu(2) Otsuka, F. L.,and Welch, M. J. (1987)Methods to Label mulation of radioactivity in the liver. Several different Monoclonal Antibodies for Use in Tumor Imaging.Nucl. Med. linkers have been investigated (21-23). The results of Biol. 14,243-249. those experiments have shown that the liver clearance (3) Hnatowich, D. J. (1990)Antibody Radiolabeling, Problems indeed depends upon the linkers, although the observed and Promises. Nucl. Med. Biol. 17, 49-55. effects were relatively small. Little is known about the (4) Liu, Y.,and Wu, C. (1991)Radiolabeling of Monoclonal mechanism of the liver metabolism of radiolabeled antiAntibodies with Metal Chelates. Pure Appl. Chem. 63,427bodies; however, several trends have been found. In 463. studying the metabolism of Lym-l-benzyl-EDTA-lllIn, (5) Subramanian, R., and Meares, C. F. (1991)Bifunctional Deshpande et al. (52) have shown that the radioactivity Chelating Agenta for Radiometal-Labeled Monoclonal Antibodies. Cancer Imaging with Radiolabeled Antibodies (D. M. excreted in the urine is still practically completely bound Goldenberg,Ed.) pp 183-199,KluwerAcademic Pubs., Boston. by the chelator. For the uptake and metabolism of ll1In(6) Wessels, B. W., and R o w , R. D. (1984) Radionuclide labeled mAb B6.2 (labeled using the cyclic DTPA anhySelection and Model Absorbed Dose Calculations for Radiodride method) by the rat liver, Jones et al. (24)found that labeled Tumor Associated Antibodies. Med. Phys. 11, 638111In seems to be trapped in the form of low molecular 645. weight In complexes (
