Bloconjugate Chem. 1991, 2, 407-414
407
Technetium Labeling of Monoclonal Antibodies with Functionalized BATOs: 2. TcCl(DMG)&PITC Labeling of B72.3 and NP-4 Whole Antibodies and NP-4 F(ab')2 K. E. Linder, M. D. Wen, D. P. Nowotnik,' K. Ramalingam, R. M. Sharkey,' F. Yost, R. K. Narra, A. D. Nunn, and W. C. Eckelman The Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, New Brunswick, New Jersey 08903, and Center for Molecular Medicine and Immunology, 5 Bruce Street, Newark, New Jersey 07103. Received May 30, 1991
BATO (boronic acid adduct of technetium dioximes) complexes, TcCl(dioxime)aBR, were prepared in which the boron substituent (R) was the protein-reactive 2-carboxy-4-phenyl isothiocyanate (CPITC). The 99Tc complexes, where the dioxime was either dimethylglyoxime (DMG) or cyclohexanedione dioxime (CDO), were prepared and characterized. The "Tc complex TcCl(DMG)&PITC was prepared from a freeze-dried kit and used to label B72.3 (anti-TAG.72) and NP-4 (anti-CEA) whole antibodies, and the NP-4 F(ab')2 fragment. SDS-PAGE electrophoresis indicated that the labeling reagent was strongly bound to antibody. The labeled antibodies displayed high binding to affinity columns and good tumor uptake in GW39 tumor-bearing mice.
INTRODUCTION In a previous publication (I), we described the preparation and protein-labeling application of a BATO (boronic acid adduct of technetium dioximes) complex functionalized with an isothiocyanato group. BATOs are seven-coordinate Tc(II1) complexes, prepared by the low pH/100 "C template reaction of a vicinal dioxime, a boronic acid, and pertechnetate (TC04-1 in the presence of the reducing agent SnClz (2,3). The ease of synthesis of BATOs from relatively simple components permitted the study of biodistribution of a large number of these neutral, lipophilic technetium complexes ( 4 ) . Two were selected for clinical evaluation as myocardial and cerebral perfusion tracers (5, 6). Addition of isothiocyanate (a functional group known to be effective in linking bifunctional chelating agents to proteins (7, 8)) to the boronic acid "cap" of a BATO transformed the chelate into a protein-labeling reagent (I). The reagent TcCl(DMG)3PITC (Figure 1)formed a stable link (presumed to be a substituted thiourea, by reaction of the isothiocyanate with the t-amino group of lysine groups in the protein) to the monoclonal antibody B72.3. Use of a preformed chelate such as a BATO is the preferred method for technetium labeling of antibodies as it eliminates the possibility of nonspecific binding of the radiotracer and, as a result, should provide a radiopharmaceutical with high in vivo stability ( 9 , I O ) . However, the low water solubility of this reagent necessitated that protein-labeling reactions be conducted in 10% aqueous DMSO. As the potential of this reagent for routine protein labeling might be increased through an improvement in water solubility, we set out to prepare and study a more water-soluble derivative of this reagent, one which contained a carboxylate group. In this paper, we describe the synthesis and characterization of the new reagent TcC1(DMG)&PITC (Figure 1)and the use of this reagent in labeling B72.3and NP-4 whole antibodies and NP-4 F(ab')2 fragments. The biodistributions of these %Tc-labeled proteins in normal and tumor-bearing mice are also described here. t
Center for Molecular Medicine and Immunology (CMMI).
R
TcCl(DMG)3PITC R=H TcCl@MG)3CPITC R=COOH Figure 1. Structures of the BATO PITC labeling reagents.
&
CH3
WH)2 10% NaOH. KMn0,
1. Mg,ether
2. (CH30)& H'
*
&Ha
RT
*
WHh
W W 2 s=C%12/3
M HCI
H2N d C 0 2 H RT * Figure 2. Synthesis of (OH)2B-CPITCS
SCN d C 0 2 H
EXPERIMENTAL PROCEDURES Materials and Reagents. Dimethylglyoxime (DMG) (Eastern Chemicals), glycine (Aldrich), and stannous chloride (MCB) were used as received. The boronic acid 3-carboxy-5-isothiocyanatophenylboronicacid [ (OH)?BCPITC] was synthesized by the five-step process shown in Figure 2. Details of the synthesis of (0H)zB-CPITC are reported elsewhere (II). The complexesqcCl(DMG)a and 99Tc(DMG)&OH)SnCl3 were prepared as described previously (3). -Tc04- in saline solution was obtained from an E.R. Squibb Minitec Generator. Freeze-dried 0 1991 American Chemical Society
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kits for the preparation of the tris-dioxime complexes mTcCl(DMG)3 and *mT~Cl(CD0)3 were manufactured as described previously (I). All other chemicals were of reagent grade and were used as received. All solvents were of either HPLC or reagent grade (except acetonitrile, which was pesticide grade) and were used as received. All water used in these studies was obtained from a Millipore Milli-Q water purification unit. B72.3 monoclonal antibody (Lot #3-178-870107, 2.81 mg/mL) was purchased from Damon Biotech. NP-4 F(ab')2 fragment and NP-4 monoclonal antibody were obtained from CMMI (NP-4is a murine IgGl monoclonal antibody that recognizes a CEA specific epitope (12)). PRP-1 resin (12-20 pm) was purchased from Alltech Associates. Sep-Pak Lite CIScartridges were purchased from Waters Associates. Most reagents and standards used in the SDS-polyacrylamide gel electrophoresis were purchased from Bio-Rad. Phast gel blue, 10-15% gradient gel, and SDS buffer strips were purchased from Pharmacia. Buffers used in the study were prepared as described previously (I). A TAG-72-Reacti-Gel affinity column in a RAININ Leur-lock column, with a 2.5-mL bed volume, was prepared in-house (1). A CEA-Affi-Gel-10 affinity column was prepared using CEA extracted from GW-39 human colonic tumor xenograft (13). Analysis of the Labeling Reagents. HPLC separations were made using a Spectra Physics Model SP8700 HPLC system equipped with an ISCO V4 UV/visible detector and a radiometric detector, dual-pen recorder, and fraction collector. The outputs from the radiation detector and spectrophotometer (set at 280 or 450 nm) were integrated by a SP4270 integrator. The data were stored and reprocessed using LABNET software. Analyses were carried out on either a 15-cm Nucleosil column (Alltech), using a mobile phase of acetonitrile/O.l M citric acid (pH 2.3) and aflow rate of 1.5 mL/min, or on a Hamilton PRP-1 column, using a mobile phase of acetonitrile/ 0.1 M NH40Ac (pH 4.6) and a flow rate of 2 mL/min. Fast-atom-bombardment mass spectra were obtained on a VG-ZAB-2F spectrometer, from a matrix of CH2Cl2/ thioglycerol. Infrared spectra were obtained on a Sirius 100 FT-IR spectrometer as KBr pellets. Proton NMR data (250 MHz) were obtained with a JEOL-GX-250 spectrometer. UV/visible spectra were measured using a Hewlett-Packard 8451A photodiode-array spectrophotometer. Elemental analyses were performed by the Squibb Microanalytical Department. Analysis of the Labeled Antibodies. HPLC methods involving the TSK-SWG3000 column (Phenomenex), the 1-cm ISRP precolumn (Alltech Associates), and a combined ISRP/TSK-SWG3000 column, Phast SDS-PAGE electrophoresis, and affinity chromatography using a TAG72-affinity column were described previously (1). Affinity chromatography on NP-Clabeled materials was conducted on a CEA-affinity column (13),using the methods described previously for the TAG-72 affinity column ( I ) . Rat serum stability studies on HPLC-purified TcC1(DMG)&PITC-labeled B72.3 were carried out as described previously for TcCl(DMG)aPITC-labeled B72.3 (I). A control sample was prepared by incubating TcCl(DMGhCPITC in PBS buffer in the absence of antibody. Both samples were incubated at 37 "C. Each sample was then evaluated by both gel electrophoresis and ISRP-TSK HPLC at 1 h after incubation. A Phast SDS-PAGE electrophoresis system (Pharmacia) was used for the characterization of 99mTc-labeled
Linder et al.
antibodies and antibody fragments using 10-15 % gradient gels and the reagents, buffers, separation protocols, and development methods recommended by the manufacturer. Synthesis of Tc Complexes. Synthesis of ggTcCl(DMG)3CPITC from TcCZ(DMG)3. To 99TcCl(DMG)3 (23.8 mg, 0.049 mmol) in 3 mL of CH3CN was added 3 drops of 1 M HCl and 11.5 mg (0.051 mmol) of (OHhBCPITC. The reaction mixture was heated at 50 "C with stirring for 15 min. It was then treated with an equal volume of 1M HC1 and cooled to room temperature. The resulting red-orange precipitate was isolated by suction filtration, washed with 15 mL of 1 M HC1, and dried in vacuo to yield 25 mg (75.6%) of relatively pure product. Recrystallization from a small volume of CH&N/ 1M HC1 yielded analytically pure needles, isolated as a 1.5-hydrate. Anal. Calcd for C ~ ~ H ~ ~ N ~ B C ~ O & TC, C 34.57; . ~ . ~ H, H~O: 3.92; N, 14.11. Found: C, 34.76; H, 3.90; N, 13.90. Synthesis of %TcCl(DMG)3CPITC from Tc(DMG)3(pcc-OH)SnC13. To *Tc(DMG)3(pcc-OH)SnC13 (84 mg, 0.114 mmol), dissolved in 15 mL of CHsCN, was added boronic acid (OH)2B-CPITC (30 mg, 0.136 mmol), followed by 1.5 mL of 3 M HC1. The reaction mixture was heated with stirring at 50 OC for 30 min. The warm solution was then treated with 40 mL of 1M HC1 and cooled to room temperature. The resulting red-orange precipitate was isolated by suction filtration, washed with 15 mL of 1 M HC1 and H20, and dried in vacuo to yield 58 mg (76%)of desired product. Following recrystallization from CHsCN/ 1 M HCl, the material thus isolated was identical to that prepared as above, as determined by TLC and HPLC (Nucleosil system, 65/35 CH3CN/O.1 M citric acid, tR = 2.85 min). Synthesis of *mT~C1(DMG)3CPITCfrom 99mTC04-. Samples of the 99mT~ complex were prepared by adding (OH)2B-CPITC (3 mg) in ethanol (50 pL) and "Tc04-/ saline (1.0 mL, 25 mCi) to a freeze-dried tris-DMG kit. The kit was heated to 70 "C for 5 min, cooled to room temperature, and passed through a Waters AssociatesSepPak Lite &cartridge. The Sep-Pak cartridge was washed with 1mL of saline and 2 mL of 25/75 EtOH/saline. The complex was eluted from the resin by treatment with 1 mL of 100% EtOH. Analysis of the reaction mixture, using the Nucleosil system above, showed the reaction mixture radiochemical purity (RCP) to be 28%. The purified material had an RCP of 60-65 % . Synthesis of %TcCl(CD0)3CPITC from TcCl(CD0)3. To 4.0 mg of TcCl(CD0)3, dissolved in 3 mL of ACN, was added 1.5 mg of (OH)2B-CPITC and 3 drops of 3 M HCl. The solution was heated gently for 20 min and monitored by HPLC at 280 nm (Nucleosil system, 60/40 ACN/O.l M citric acid. During this time, TcCl(CD0)3 (tR = 5.0 min) and the boronic acid (tR= 1.66min)both disappeared, and a peak at tR = 8.0 min formed. The UV/visible spectrum of this reaction mixture in acetonitrile indicated that a BAT0 complex had formed (maxima at 276,386, and 466 nm). When analyzed by FAB mass spectroscopy, a strong molecular ion at the expected mass of TcC1(CDO)&PITC ( m / e 745/747) was observed in both positive and negative modes. Synthesis of mT~Cl(CD0)3CPITC. Method I from 99mTco4-. To a freeze-dried tris CDO kit was added (OH)2B-CPITC (3 mg) in ethanol (50 pL) and wmTc04-/saline (1.0 mL, 25 mCi). The kit was heated at 70 "C for 5 min and purified on a Waters Sep Pak Lite C-18 cartridge as described above for 99mTcC1(DMG)&PITC. The RCP of the reaction mixture before purification was 15%;after purification the RCP was 67 % . Method II: from 99mT~C1(CD0)3. A sample of WmTcCl-
Technetlum Labeling of Monoclonal Antibodies
(CDO)3was prepared by adding 24 mCi of TcOr- in 1mL of saline to a freeze-dried kit containing CDO. After heating at 100 "C for 5 min, the RCP of mT~C1(CD0)3 was 92%. To this kit was added (0H)pB-CPITC (2.4 mg) in ethanol (50 pL). The mixture was heated for 15 min at 100 "C to give 99mT~C1(CD0)3CPITC in 49 % yield. After purification as described above, the RCP was 74 % . Method ZZI from ggmT~C1(CD0)3.A sample of -TcCl(CD0)3 was prepared as described in method I1 above. After heating at 100 "C for 5 min, the RCP of -TcCl(CD0)3 was 92 % . The TcCI(CD0)3 was isolated, and kit components were removed, by purification on Hamilton PRP-1 resin. The kit contents were drawn up into a needle-hub of resin and then rinsed with 1 mL of saline, 1 mL of 25/75 EtOH/saline, 1 mL of Hz0, and 1 mL of EtOH. The ethanol fraction contained 17 mCi of TcCl(CD0)3 (RCP = 86.6%). Half of the ethanol fraction was added to a vial that contained 1.5 mg of (OH)2BCPITC and 50 pL of 1N HC1. This kit was heated for 5 min at 100 "C and then reanalyzed by HPLC as described above. The yield of TcCl(CD0)3CPITC prepared by this method was 82 % (94% yield based on the purity of starting TcCl(CDO)3. Sep-Pak Lite purification as described above did not increase the RCP. T h e Effects of pH,Temperature,and Heating Time on t h e RCP of ggmTcC1(DMG)3CPITC. 9gmTcC1(DMG)&PITC was prepared by adding (OH)2B-CPITC (3 mg) in ethanol (50 pL) and mTc04- (1.0 mL, 20-30 mCi) to a freeze-dried kit containing DMG. The effects of heating temperature (70-100 "C), heating time (5-50 min), and pH (2.0-4.0) on the RCP of the labeling reagent were evaluated as was described for TcCl(DMG)3PITC in ref 1. S t u d i e s of t h e Reactivity of t h e NCS Group. Reaction of (0H)zB-PITC and (OH)&-CPITC with Glycine. Equimolar solutions of (OH)2B-PITC (I) and (0H)zB-CPITCwere made by dissolving 4.80 mg (0.0268 mmol) or 6.00 mg (0.0269 mmol) of ligand, respectively, in 4.0 mL of EtOH. An 0.01385 M glycine solution was prepared by dissolving 10.4 mg of glycine in 10.0 mL of 0.1 M sodium phosphate buffer (pH 9.5). All three solutions were brought to 37 "C. At t = 0 , l mL of boronic acid solution (0.0067 mmol) was added to 4 mL of glycine solution (0.0553mmol, 8.3-fold excess). The mixture was incubated at 37 "C. At 5 min-intervals, 25-pL aliquots from both reaction mixtures were removed and analyzed by HPLC, using a Nucleosil C8 column and CH&N/O.l M citric acid mobile phase at 1.5 mL/min. The solvent ratio for samples from the reaction with (H0)2B-PITC was 40/60, while, for the (0H)zB-CPITC reaction, a 30/ 70 CHaCN/citric acid mixture was used. The disappearance of the boronic acid was determined by the change in area under the boronic acid peak at 290 nm. Hydrolysis of 99TcCl(DMG)3CPZTC to g9TcOH(DMG)3CPZTC. A sample of 10 mg of 99TcCl(DMG)3CPITC was dissolved in 5 mL of CH3CN and 5 mL of 0.1 M sodium phosphate buffer (pH 9.5). The apparent pH of this mixture was 9.0 on wet E. Merck narrow-range pH paper. The solution was incubated at 37 "C ( f l "C) for 100 min. During this time, the color of the reaction mixture changed from red-orange to yellow-orange. Aliquots of the reaction were analyzed by HPLC on a Nucleosil column that was eluted with 65/35 CH&N/O.l M citric acid at a flow rate of 1.5 mL/min. TcCl(DMG)&PITC (tR = 2.89 min) was cleanly converted to a more polar complex (TcOH(DMG)&PITC) at t~ = 1.86 min, as detected by absorbance at 280 nm. After 70 min, an aliquot of the mixture was acidified to
BioconJugate Chem., Vol. 2, No. 6, 1991 409
pH 1.0, by treatment with 2 drops of concentrated HC1. The original color returned immediately. HPLC analysis of this solution showed almost full conversion back to starting material. Aqueous Solubility of 99mT~C1(DMG)3CPITC. 99"TcCl(DMG)&PITC was prepared by heating at 100 "C for 5 min and purified as described above. The purified labeling reagent in ethanol was divided into three 300-pL aliquots. Each sample was dried under a stream of Nz. An aliquot of 100 pL of EtOH, 100 pL of 0.1 M sodium phosphate buffer (pH 9.5), or 5 pL of DMSO in 95 pL of pH 9.5 phosphate buffer was added to the dried m T c complex. The samples were then incubated at room temperature for 2 h, after adding an additional 100 pL of phosphate buffer (pH 9.5) to each vial. The sample in each vial was transferred into a separate test tube after incubation. The radioactivity recovered in the test tube and the radioactivity remaining in the vial were determined with a dose calibrator. The percent recovery was calculated as radioactivity recovered/ (radioactivity recovered + radioactivity remaining in the vial). General Procedure for Labeling of Monoclonal Antibodies a n d Fragments w i t h 99mT~Cl(DMG)3CPITC. A 300-pL aliquot of the purified BmTcC1(DMG)&PITC solution in ethanol was dried under a stream of Nz and 100 pL of the antibody or antibody fragment (10 mg/mL in 0.1 M sodium phosphate buffer, pH 9.5) was added to the dried B m Tcomplex. ~ The reaction mixture was incubated at 37 "C for 2 h. The B m Tlabeled ~ antibody was then purified using an ISRPTSK HPLC system, as described previously (I). Biodistribution Studies. I n Normal Mice. The biodistributions of 99mT~C1(DMG)3CPITC and of BmTcC1(DMG)&PITC-labeled B72.3 whole antibody, -labeled NP4 F(ab')z, and -labeled NP4 whole antibody were evaluated in normal mice (n = 4 or 5/study). Mice were injected iv with about 1 pCi (100 pL) of either the m T c complex or the BmTc-labeled antibody and sacrificed at 1 or 24 h after injection. Organs of interest were taken, weighed, and assayed for radioactivity. The percent injected dose/organ and percent injected dose/gram of tissue were determined. I n Tumor-Bearing Mice. GW39 tumors (14,15) were grown serially in nude mice by inoculation with 0.2 mL of a 10% tumor suspension. The suspension was prepared by mincing an established tumor and then passing this preparation through a 40-mesh wire screen to obtain a uniform cell suspension. After 14 days, 10-20 pCi of labeled antibody (B72.3, NP-4, or NP-4 F(ab')z fragment) was administered via tail vein injection. Mice (n = 4-8 for each time point) were dissected at 1or 24 h after injection. For the studies with Tc-labeled B72.3, a sample of 1311-B72.3 (prepared by chloramine T iodination) (16)was injected into another group of tumor-bearing mice as a control. The percent injected dose/organ ar.d percent injected dose/gram of tissue were determined.
RESULTS AND DISCUSSION Synthesis and Characterization of Wc Complexes. As was the case for TcCl(DMG)3PITC (I), gSTcC1(DMG)&PITC can be prepared in good yield from either C~~. TcCl(DMG)3or T C ( D M G ) ~ ( P - O H ) S ~gsTcCl(CD0)3CPITC was prepared quantitatively from TcCl(CD0)3. These compounds are expected to have a structure that is quite similar to our first-generation protein-labeling reagent, TcCl(DMG)3B-PITC, which was characterized by X-ray crystal structure analysis (I). All analytical data
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Bioconlugate Chem., Vol. 2, No. 6, 1991
Table I. HPLC Retention Times for TcCl(DMG)&PITC and Important Side Products solvent buffer complex column' ratio pH t R , min 40/60 4.6 13.0 TcCl(DMG)3CPITC PRP-1 40/60 5.1 2.2-2.5' TcCI(DMG)&PITC PRP-1 65)35 4.6 3.1-3.5' TcCl(DMGjjCP1TC PRP-1 50/50 4.6 6.2-10' TcCI(DMG)3CPITC PRP-1 2.3 2.85 Cg 65/35 TcCl(DMG)3CPITC 50/50 4.6 1.45 TcOH(DMG)&PITC PRP-1 2.3 1.85 Cg 65/35 TcOH(DMG)&PITC PRP-1 50/50 4.6 6.8 TcCl(DMG)3 2.3 2.67 C8 60/40 TcCl(DMG)3 0 For the PRP-1 system, the mobile phase solvent mixture is ACN/ 0.1 M NHdOAc at 2 mL/min. Note (I): the retention time is variable using this buffer, being highly dependent upon the pH of the mobile phase. For the Nucleosil Cg system, the mobile phase is ACN/O.l M citric acid, at 1.5 mL/min.
on the 99TcCl(dioxime)3CPITC complexes suggests that this similarity exists. The 'H NMR spectrum of TcCl(DMG)&PITC is as expected. Phenyl resonances for the trisubstituted phenyl boron cap are seen at 7.2-7.8 ppm. The methyl resonances for the two equivalent dimethylglyoxime ligands and the one unique ligand fall between 2.2 and 2.4 ppm. The two oxime protons (0--H- -O),which are seen at 15.1ppm in the spectrum of the PITC complex, are not seen in the spectrum of the CPITC compound, nor is a peak for the carboxylic acid proton (COOH). However, the large water peak in this spectrum (the complex was isolated as a hydrate) suggests that the disappearance of these resonances is due to exchange with water. The FAB +/- mass spectra of 99TcC1(DMG)3CPITC show strong molecular ion peaks at m/z 668/670 (M + H)+ or 666/668 (M - H)- in the positive and negative modes, respectively. Losses observed from these ions parallel fragmentation patterns observed previously in the spectra of several other BATO complexes (17). The infrared spectrum of TcCl(DMG)3CPITC contains a pronounced peak at about 2100cm-l due to the NCS group; strong carboxylate absorptions are seen at 3440 (vs) and 1720 cm-' (m). The retention time of TcCl(DMG)&PITC on reversedphase HPLC is very sensitive to the buffer pH (Table I). Changing buffer pH from 4.6 to 5.1 results in a change in the HPLC retention time from 13 to 2.5 min. This is consistent with a large change in the degree of ionization of the carboxylic acid functionality on the boronic acid cap, from primarily free acid at pH 4.6 to partially deprotonated form at pH 5.1. Coinjection studies withTcC1(DMG)&PITC and 99mT~Cl(DMG)3CPITC were carried out in several HPLC systems (the systems shown in Table I). Coelution of the two compounds was noted in all cases. Synthesis of 99mT~ Complexes. When the SmTcC1(dioxime)aCPITCcomplexes were prepared directly from pertechnetate, yields were very poor (90% are regularly obtained (6, 18). Attempts to improve the yield of the CPITC complexes by varying pH, temperature, and reaction time were relatively unsuccessful. Although these factors had an effect on yield that was similar to those reported previously for the PITC complex, the optimum yield obtained for 99mTcC1(DMG)3CPITC was only 28% when heated at 100 "C for 5 min at pH 2.0. At longer reaction times, considerable degradation of the labeling reagent was seen (Figure 3). Under similar
Linder et al.
q
i\
20
% RCP
l2I 10
I pH 2; 108C I
0
10
5
15
20
25
healing time (minutes)
Figure 3. The influence of reaction time on the RCP of 99mTcCl(DMG)3CPITC(pH = 2.0 at 100 "C). Table 11. Comparison of the Recovery of 'DmTcC1(DMG)sPITC and wmTcC1(DMG)aCPITC ?6 recovery from viala
activity dissolved in 50% EtOH/50% PO,- buffer 5% DMS0/95% Pod- buffer 100A Pod- buffer
TcCl(DMG)sPITC 20 59 10
TcCl(DMG)SCPITC 92.6 90 89
Percent recovery from vial following a 1-h incubation at 37 OC in the specified solvent.
conditions, the yield of 99mT~C1(CD0)3CPITC was only 15% at 5 min, when prepared directly from pertechnetate. The yield of 99TcCl(DMG)3CPITC was also low when prepared directly from %Tc04-, but good when prepared from either of the intermediates in BATO formation (3), Tc(dioxime)3&-OH)SnC4or Td=l(dioxime)a. These trends led us to consider whether kit components were responsible proteinfor the poor radiochemical yields of the 99mT~ labeling reagents when prepared directly from pertechnetate. A study was conducted in which the BATO precursor 9%TcCl(CD0)3 was prepared from a kit, and then the boronic acid cap was added to the reaction mixture. The yield in this reaction (method 11) was increased to 49 % (compared to a 15% yield when prepared directly from pertechnetate). When the 9SmTcC1(CDO)awas isolated from all kit components by solid-phaseextraction and then mixed with boronic acid at pH 2.0 (method 111),the desired BATO complex could be prepared in 8 2 % yield (equivalent to 94 % conversion based on purity of 99"TcC1(CD0)3starting material). Thus, it is clear that one of the kit components, and not low pH or heating, is responsible for the poor yield of these protein-labeling reagents when prepared directly from Tc04-. The implication is that the proteinlabeling BATOs can be made in a reasonable overall yield but that this improvement in (50-60s) from 99mT~04-, yield is achieved through an additional solid-phase purification step. Aqueous Solubility of WmTcCl(DMG)3CPITC. Addition of a carboxyl group to the PITC cap was intended to improve the water solubility of the protein-labeling reagent above that previously observed with "TcCl(dioxime)sPITC. This appears to have been achieved. The solubilities of 99mTcCl(DMG)3PITCand TcC1(DMG)&PITC in a variety of solvents are compared in Table 11. Of note is the fact that after a 1-h incubation of 99mTcCl(dioxime)3PITC in phosphate buffer, recovery was only 10% (the remainder remaining adhered to the vial wall), compared with 89% recovery for *TcCl(dioxime)&PITC. Thus, protein labeling with the CPITC
Bloconjugate Chem., Vol. 2, No. 6, 1991 411
Technetium Labeling of Monoclonal Antibodies
reagent may be conducted without the addition of an organic modifier to solubilize the reagent. The carboxylic acid substituent is presumably in the ionized form at the pH (9.0) at which protein labeling occurs, creating a complex with a net 1- charge. This conclusion is drawn from HPLC data, where it is observed that complex retention time varies significantly in the pH range 4.65.0, indicating that the apparent pK, of the carboxylic acid is close to this range ( I S 2 I ) . The presence of an ionized group on the phenyl ring is expected to alter the reactivity of the isothiocyanate electrophile toward proteins (compared with PITC, the ligand without a carboxylate group), although the meta orientation of carboxylate to isothiocyanate should minimize this effect (22). For this reason, various studies were undertaken to examine the reactivity of CPITC compared to that of PITC. Hydrolysis of Labeling Reagent in the Absence of Protein: 99TcCl(DMG)aCPITC to 99TcOH(DMG)3CPITC. Hydrolysis of TcCl(DMG)3CPITC at pH 9.5/ 37 "C gave a 95% yield of a more polar product, accompanied by a color change from red-orange to yelloworange. However, on treatment with HC1 (pH = l.O), the original color of the reaction mixture returned immediately. HPLC analysis of the acidified solution showed almost full conversion back to TcCl(DMG)3CPITC. These results indicate that hydrolysis of the NCS group did not occur under these conditions, as NCS hydrolysis to the amine is not reversible under the conditions used. The reaction observed is consistent with the product of hydrolysis being TcOH(DMG)&PITC, where the axial chloride ligand has been replaced a hydroxide. Chloro/ hydroxy exchange in BATOs is known (23) to occur at a rate that is consistent with the time course observed. Similar results were observed with both 99mTcC1(DMG)3PITC and 99mTcC1(DMG)3CPITC.Both complexes were hydrolyzed in pH 9.5 phosphate buffer (10% DMSO) to more polar products within 2 h. The HPLC retention time of the hydrolysis product of 99mTcC1(DMG)&PITC was identical to that of authentic99TcOH(DMG)3CPITC. Based on these results, we expect that chloro/ hydroxy exchange to form *TcOH(DMG)3CPITC also occurs in protein labeling reaction mixtures. However, the nature of the axial ligand on the BATO is unlikely to have much influence on the performance of a derivatized BATO as a protein-labeling reagent (except to improve solubility; hydroxy BATOs are much less lipophilic than their chloro analogues (24)). The significant result is that little hydrolysis of the isothiocyanate group takes place under protein-labeling conditions for either reagent. Reactions of PITC and CPITC Reagents with Glycine and Antibodies. When the boronic acids (OHhBPITC and (OH)2B-CPITC were incubated with glycine under identical conditions, coupling of the NCS group on the boronic acid and glycine occured and both boronic acids disappeared (Figure 4). The reaction half-lives were 18min for (0H)zB-PITCand 22.8 min for (OH)2B-CPITC, indicating similar reactivity toward amines. This result indicates that all other things being equal, the PITC and CPITC reagents should be equally reactive toward proteins. However, in the labeling of B72.3 antibody, the BATO with the water-solubilizing carboxylate function proved to be markedly superior. On the basis of five experiments, the antibody labeling yield is higher for *TcCl(DMG)3CPITC than it is for *TcCl(DMG)3PITC (32.4%, compared to 7.5 % ). These yields are lower than that observed for reaction with glycine, presumably due to the lower concentration of antibody (0.2 mM) compared
100 80
Pu
.. remaining
4o
20
l0 o
b 0
5
10
15
20
25
30
35
40
45
50
lime (minules)
Figure 4. Graph showing the disappearance of the isothiocyanate reagents on reaction of (H0)2B-PITC and (H0)zB-CPITC with glycine.
to glycine (10mM)and the lower accessibilityof the reagent to protein €-amino lysine groups compared to the amine group on glycine. We have shown previously ( 1 )that there is no binding of BATO to protein using a BATO complex which did not contain an isothiocyanate group, so that these protein binding yields should reflect specific binding only. Following isolation of the labeled antibodies by HPLC, it was found that both protein recovery and immunoreactivity of the product were also slightly better for B72.3 labeled with 9gmTcC1(DMG)3CPITCthan with "TcC1(DMG)3PITC (71.4% and 67 % protein recovery and 72% and 60% binding to TAG-72 affinity columns, respectively). Labeling studies with 99mT~C1(DMG)3CPITC were then extended to examine the radiolabeling of the NP-4 whole antibody, and the NP-4 F(ab'):! fragment. Percent incorporation of radiolabel, protein recovery, and immunoreactivity results are given in Table 111. NP-4 is an anti-CEA IgG, which has been compared previously to B72.3 in the nude mouse model (25). The radiolabeling yields and protein recoveriesfor NP-4 whole antibody were similar to those obtained for B72.3. Immunoreactivity studies on isolated NP-4 labeled with -TcCl(DMG)3CPITC showed little binding to a TAG-72 affinity column (which contains an irrelevant antigen that does not bind to NP-4), but >90% binding to a CEA affinity column (Table 111). The labeling efficiency and protein recovery of NP-4 F(ab')2 labeled with ggmTcC1(DMG)3CPITCwere lower than for the whole antibody. As the concentration of NP-4 antibody and its F(ab')z fragment were similar in these labeling experiments, the reason for this difference is uncertain at this time. Characterization of Labeled Antibodies. As in our previous study ( I ) , the stability of the link between label and antibody was demonstrated by SDS-PAGE. Under nonreducing conditions, 90.6% (B72.3) and 82% (NP-4 whole antibody) of the radioactivity was found to be associated with antibody. Reduction with P-mercaptoethanol resulted in significant loss of the label, observed as activity at the dye front in gels that were not subjected to the stain/destain protocol (destaining washes this dyefront activity away). F(ab'):! fragments, under nonreducing conditions, migrate in SDS-PAGE to a single band at 100 kDa; 88% of total radioactivity was associated with this fraction in the nonreduced labeled fragment. When reduced with p-mercaptoethanol, F(ab'):! fragments give a doublet of bands at 25 kDa on SDS-PAGE. The lower band of the doublet corresponds to the light chain, while the higher band is the amine-terminal portion of the cleaved heavy chain
412
Llnder et
Bioconjugate Chem., Vol. 2, No. 6, 1991
Table 111. Radiolabeling of B72.3 and NP-4 with 00”I’cCl(DMG)&PITC: protein B72.3 whole antibody NP-4 whole antibody
protein B72.3 whole mAba NP-4 whole mAb NP-4 F(ab’)z
Yield and Characterization of Products
a. Yield and Immunoreactivity (Affinity Column) SO labeling 9; protein recovery 32 71 26 75
NP-4 F(ab’)z
15
gel not stained gel stained/destained gel not stained gel stained/destained gel not stained gel stained/destained
al.
43
?6 immunoreactivity
72” 6‘ 96b 93b
b. SDS-PAGE unreduced: reduced with 8-mercaptoethanol Oio bound to whole mAb % bound to the heavy chain % bound to the light chain 90.6 35.7 8.5 97 84 10.2 82 18 9 96 64 18 88 43‘ 80 83‘
a Binding to the TAG-72 affinity column (irrelevant antigen for NP-4 antibody). Binding to the CEA affinity column. Light chain plus NH2-terminal portion of the cleaved heavy chain (Fd).
Table IV. Comparison of the Biodistribution of mTcC1(DMG)sPITC, 99mTcC1(DMG)&PITC,and Whole B72.3 Labeled with These Reagents, in Normal Mice
blood heart lungs brain liver spleen GI kidneys muscle
blood heart lungs brain liver spleen GI kidneys muscle
lh 0.42 4.33 3.52 0.99 1.03 11.0 42.49 2.23
lh 30.33 6.90 8.57 1.06 8.19 2.44 5.61 1.00
Distribution of Labeling Reagents in Normal Mice %ID/g in tissue =TcCl(DMG)3PITC, n = 5 *TcCl(DMG)&PITC, n = 4 SD 24 h SD lh SD 24 h 0.24 2.29 1.12 7.43 4.82 2.85 1.54 3.14 0.67 0.21 1.84 0.35 1.38 1.00 0.34 2.78 1.04 1.05 0.12 0.39 0.11 0.11 0.32 0.14 0.18 3.62 0.24 4.92 1.20 0.98 1.37 0.53 0.63 1.16 1.80 0.66 22.20 1.10 0.32 1.06 0.98 0.12 0.15 2.83 0.32 0.98 0.21 0.15 1.31 1.15 0.33 Distribution of Tc(C)PITC-Labeled B72.3 in Normal Mice % ID/g in tissue *TcCl(DMG)sPITC-B72.3, n = 5 *TcCl(DMG)&PITC-B72.3, n = 4 SD 24 h SD lh SD 24 h 5.12 13.09 1.95 29.61 5.50 13.37 2.10 3.51 1.35 7.14 1.07 6.27 4.39 1.48 13.15 3.29 2.86 4.99 0.51 0.51 1.02 1.06 0.74 0.57 4.19 0.34 0.87 8.06 5.63 0.69 2.31 0.29 4.50 0.79 4.83 2.91 0.41 0.16 2.59 0.24 1.79 2.76 1.28 6.17 0.56 4.48 0.78 2.36 0.78 0.24 1.45 1.92 0.18
(Fd)(26). Only43 ?4 of the original activityremains bound to these reduced fragments. Serum Stability of B72.3 Labeled with mTcC1(DMG)&PITC. The stability of the radiolabel in serum was demonstrated by incubation of B72.3 labeled with BmTcCl(DMG)3CPITCin rat serum; no loss of label was observed after 1 h in serum. Only one radioactive component, coincident with the radiolabeled monomer, could be detected on analysis of the labeled antibody in rat serum (1 h at 37 “C) by ISRP-TSK HPLC. By electrophoresis, 96.1 $;i of total radioactivity in rat serum was associated with the whole antibody fragment. No activity was found to be bound to any serum component. In a control study, in which B72.3-labeled with 99mTcC1(DMG)&PITC was incubated in buffer for 1h, 96.2% of the radioactivity was associated with the protein monomer. Biodistributions. Biodistribution data are given in Tables IV-VI. Table IV provides a comparison of the biodistributions in normal mice of the labeling reagents
SD 1.20 1.40 0.31 0.02 0.10
0.26 0.06
0.16 0.26
SD 1.44 2.02 1.39 0.09 1.14 2.17 0.57 0.83 0.80
BmT~Cl(DMG)3PITC and wmTcC1(DMG)3CPITCand of whole B72.3 labeled with these reagents. Data on the biodistributions of B72.3 in GW39 tumor-bearing mice, labeled with either 99”TcC1(DMG)3CPITCor 1311,aregiven in Table V. Biodistribution data on TcCl(DMG)&PITClabeled NP-4 (whole antibody and F(ab’)a fragment) in normal and GW39 tumor-bearing mice are given in Table VI. The biodistribution of the labeling reagents BmTcC1(DMGIBPITCand BmTcC1(DMG)3CPITCare very different from one another. In normal mice, the neutral PITC complex shows rapid clearance through the kidneys; the anionic CPITC compound appears to clear through the GI system. In contrast, the normal mouse distributions of B72.3 labeled with these two reagents are quite similar to one another, and very different from that of either labeling reagent. Both Tc reagents have less than 3% ID/g in blood at 24 h. When the antibody B72.3 is labeled with either compound, blood values (30 and 137% ID/g at
Bioconjugate Chem., Vol. 2, No. 6, 1991 413
Technetlum Labeling of Monoclonal Antibodies
Table V. Comparison of the Biodistributions in GW39 Tumor-Bearing Mice of B72.3 Labeled with either mTcC1( DMG)aCPITC or l*lI % ID/g in tissues at times in tumor-bearing mice, n = 5 wmT~-B72.3 lh
blood liver GI stomach kidneys urine spleen muscle bone thyroid tumor
30.66 9.05 4.47 2.51 10.49 7.88 7.70 1.37 2.91 8.94 2.48
SD 3.36 0.72 0.52 1.95 3.70 0.90 1.08 0.24 0.59 3.43 0.75
13'I-B72.3
24 h 15.84 6.13 3.31 1.60 5.17 6.50 3.82 1.46 1.91 4.79 10.47
SD 2.26 1.23 0.30 0.34 0.87 3.64 1.10 0.32 0.41 2.04 3.79
lh
25.96 7.80 2.04 2.46 11.19 8.45 8.57 1.33 3.43 27.65 3.10
SD 7.32 1.17 0.43 0.72 6.66 4.89 3.17 0.44 1.21 12.10 1.41
24 h 18.98 5.50 1.45 4.47 6.26 4.28 5.11 2.16 2.63 45.95 18.20
SD 2.71 1.13 0.18 1.12 1.03 1.70 1.51 0.32 0.48 14.67 8.36
Table VI. Biodistribution of Labeled NP-4 (Whole Antibody and F(ab')z Fragment) in Normal and GW39 Tumor-Bearing Mice % ID/c in tissue normal mice hTeNP4F(ab')z l h 24 h blood heart lungs kidneys liver tumor
tumor-bearing mice 99mT~NP4F(ab')z 2h 24 h
hT~-NP4 l h
24 h
24.3 3.4 5.9 7.8 3.3
2.9 1.7 1.9 5.8 1.0
27.5 6.3 8.3 4.9 8.1
17.0 4.0 5.1 3.9 5.2
n=5
n=5
n=5
n=5
1and 24 h, respectively) are dramatically higher than those observed for the labeling reagents themselves. These high blood values are in keeping with the slow blood clearance that is expected for a labeled antibody. These data strongly suggest that the link between the labeling reagents and protein is strong and that substantial loss of label does not occur. This is substantiated by a comparison of the biodistributions in tumor-bearing mice of B72.3 labeled with either 1311or TcCl(DMG)&PITC (Table V). There are no significant differences in the organ distributions of the two labeled proteins, except that the GI values are slightly higher for the Tc-labeled protein and the thyroid values are substantially higher for the iodinated antibody. Tumor uptake for the iodine-labeled B72.3 was higher, but not significantly so. Substantial variation in 5% ID/g in the tumors was seen, with the highest ?4 ID seen in the smallest tumors. This pattern has been noted previously by others (27-29). The biodistribution of TcCl(DMG)&PITC-labeled NP-4 in both normal and tumor-bearing (nude) mice was also performed. NP-4 is a monoclonal antibody that is specific for carcinoembryonic antigen (CEA) and which, unlike some B72.3 monoclonals, does not significantly complex to circulating blood CEA. The tumor uptake of Tc-CPITClabeled B72.3 and NP-4 were similar at 24 h (10.5 f 3.8% vs 16.6 f 5.8% ID/g, respectively), but because blood values for labeled NP-4 were lower at 24 h, the tumor to blood ratio was improved from 0.7 to 1.7. Because imaging at such low tumor to blood ratios is not feasible, and because the short half-life of BmTc precludes imaging at later times, we have also briefly explored the use of F(ab')P fragments labeled with TcCl(DMG)&PITC. It is well-known that blood clearance for such fragments is much faster than that observed with whole antibodies (9). As shown in Table VI, blood clearance for the Tc-labeled fragment was significantly faster than that of whole NP-4. However, absolute tumor
hTc-NP4 2h
24 h
29.4
6.2
21.0
11.5
10.9 17.7 8.0 4.0 n=4
3.8 18.0 3.3 10.5 n=8
10.5 7.1 5.8 1.8 n=6
5.4 3.5 4.3 16.6 n=6
uptake was also somewhat lowered, so an improvement in tumor to blood ratios was not noted at 24 h. Significant activity was, as expected, found in the kidneys when Tclabeled NP-4 fragment was used. The distributions observed above are those expected of radiolabeled antibodies and are very different from those of the labeling reagent itself, suggesting again that the Tc-protein linkage in these BATO-labeled antibodies is a stable one. However, despite the fact that the BAT0 labeling reagent TcCl(DMG)&PITC can be prepared in good yield, and incorporation of the CPITC complex into proteins was substantially improved over that seen with our previous PITC reagent (30% vs 7.5% labeling), the overall yield of Tc-labeled protein is still disappointingly low. It appears that the reactivity of the isothiocyanate group toward amines under the protein-labeling conditions used may not be sufficient. Further work should probably focus on the use of other, more reactive linking groups. ACKNOWLEDGMENT
We thank Lillian Belnavis, Mary Ann Homack, and Christine Hood for technical assistance with the biodistribution studies in normal mice at Bristol-Myers Squibb and Rosarito Aninipot for technical assistance with the biodistribution studies in tumor-bearing mice at CMMI. LITERATURE CITED (1) Linder, K. E., Wen, M. D., Nowotnik, D. P., Malley, M. F., Gougoutas, J. Z., Nunn, A. D., and Eckeiman, W. C. (1991) Technetium-labeling of monoclonal antibodies with functionalized BATOs: 1. TcCl(DMG)3PITC. Bioconjugate Chem. 2, 160-170. (2) Treher, E. N., Francesconi, L. C., Gougoutas, J. Z., Maliey, M. F., and Nunn, A. D. (1989) Monocapped tris(dioxime) complexes of technetium(II1): Synthesis and structural characterization of TcX(dioxime)sB-R(X = C1,Br;dioxime = dimethylglyoxime, cyclohexanedione dioxime; R = CH,, CIHs). Znorg. Chem. 28, 3411-3416.
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Registry No. @@TcCl(DMG)3, 127709-21-1; (OHIgB-CPITC, 136537-21-8; @@TcCl(DMG)3CPITC, 136537-22-9; T c ( D M G ) ~ (pOH)SnC13, 127686-39-9; 9@mTcO4-,23288-61-1; s s T ~ C l (CDO)&PITC, 136537-23-0; @@TcCl(CDO)s, 127686-41-3; TCOH(DMG)sCPITC,136586-68-0;(OH)*B-PITC,133887-74-8;glycine, 56-40-6.
