Radiobromination of humanized anti-HER2 monoclonal antibody trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate, a potential label for immunoPET

Nuclear Medicine and Biology 32 (2005) 613 – 622 www.elsevier.com/locate/nucmedbio

Radiobromination of humanized anti-HER2 monoclonal antibody trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate, a potential label for immunoPET Eskender Mumea, Anna Orlovab, Per-Uno Malmstrfmc, Hans Lundqvistd, Stefan Sjfberga, Vladimir Tolmachevd,T a

Organic Chemistry, Department of Chemistry, Uppsala University, S-751 24 Uppsala, Sweden b Affibody AB, S-161 02 Bromma, Sweden c Division of Urology, Department of Surgical Sciences, Uppsala University, S-751 85 Uppsala, Sweden d Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, S-751 85 Uppsala, Sweden Received 3 March 2005; received in revised form 17 April 2005; accepted 17 April 2005

Abstract Combining the specificity of radioimmunoscintigraphy and the high sensitivity of PET in an in vivo detection technique could improve the quality of nuclear diagnostics. Positron-emitting nuclide 76Br (T 1/2 = 16.2 h) might be a possible candidate for labeling monoclonal antibodies (mAbs) and their fragments, provided that the appropriate labeling chemistry has been established. For internalizing antibodies, such as the humanized anti-HER2 monoclonal antibody, trastuzumab, radiobromine label should be residualizing, i.e., ensuring that radiocatabolites are trapped intracellularly after the proteolytic degradation of antibody. This study evaluated the chemistry of indirect radiobromination of trastuzumab using N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate. Literature data indicated that the use of this method provided residualizing properties for iodine and astatine labels on some antibodies. An optimized bone-potQ procedure produced an overall labeling efficiency of 45.5F1.2% over 15 min. The bromine label was stable under physiological and denaturing conditions. The labeled trastuzumab retained its capacity to bind specifically to HER2-expressing SKOV-3 ovarian carcinoma cells in vitro (immunoreactivity more than 75%). However, in vitro cell test did not demonstrate that the radiobromination of trastuzumab using N-succinimidyl 5-bromo-3pyridinecarboxylate improves cellular retention of radioactivity in comparison with the use of N-succinimidyl 4-bromobenzoate. D 2005 Elsevier Inc. All rights reserved. Keywords: Indirect radiobromination; Monoclonal antibody; N-Succinimidyl 5-bromo-3-pyridinecarboxylate

1. Introduction Radionuclide tumor targeting uses molecular recognition of tumor-associated structures for selectively delivering radionuclides to tumors for diagnostic or therapeutic purposes. A number of radiolabeled antibodies or antibody products are in routine clinical use, providing excellent specificity in tumor diagnostics. It should be noted that the radionuclides routinely used for targeting, 111In and 99mTc, as well as an actively evaluated 123I, are single-photon emitters, which predetermines the use of single-photon detection techniques, such as gamma scintigraphy or SPECT. Despite impressive achievements in diagnostic T Corresponding author. Tel.: +46 18 471 34 14; fax: +46 18 471 34 32. E-mail address: [email protected] (V. Tolmachev). 0969-8051/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2005.04.010

instrumentation and algorithms, intrinsic limitations of those methods, such as low registration efficiency and low resolution, still restrict the sensitivity of radioimmunoscintigraphy, especially in detecting small tumors. One way to improve radioimmunodetection sensitivity is to use positron emission tomography (PET) as an in vivo detection technique. Positron emission tomography is superior to single-photon detection in terms of registration efficiency and resolution [1]. However, a major obstacle to the use of PET for radioimmunodiagnostics is the short half-lives of conventional positron emitters, such as 11C (T 1/2 =20.4 min) or 18F (T 1/2 =109.8 min). Given that targeting molecules often have distribution times of several hours to days, conventional PET nuclides are not applicable, and alternative positron-emitting nuclides with matching half-lives and with suitable labeling properties,

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such as, e.g., I (T 1/2 =4.18 days), 89Zr (T 1/2 = 3.27 days), 55 Co (T 1/2 =17.53 h) or 76Br (T 1/2 =16.2 h), are thus necessary [1,2]. One possible long-lived positron-emitting label for mAbs and their fragments is 76Br. This nuclide has a half-life of 16.2 h and decays by emitting positrons (54%). The use of the 76 Se(p,n)76Br nuclear reaction and enriched Cu2Se targets enables the production of useful quantities of 76Br using 16-MeV cyclotrons, which are available at many PET centers [3]. Alternatively, this nuclide could be used in a satellite PET center in combination with regional production [4]. The imaging and quantification properties of 76Br have been carefully studied [5,6]; it has been demonstrated that despite its relatively high positron energy (E h+max = 3.9 MeV), resolution degradation is minor compared to that of 18F. Our group has studied the radiobromination of antitumor mAbs and their biological behavior for some years [5,7–10]. Poor cellular retention and poor excretion of bromide (the major radiocatabolite of directly radiobrominated mAbs) from organisms led to reduced imaging contrast and were identified as major obstacles to their clinical use. The use of N-succinimidyl-para-bromobenzoate [10] solved the problem of poor clearance of bromide from extracellular space (Tolmachev, unpublished results), but the problem of poor cellular retention remains to be resolved. The literature suggests that the most probable reason for poor tumor retention of radiohalogens is the leakage of lipophilic radiocatabolites through lysosomal and cellular membranes after cellular processing and degradation of antibodies [11]. Logically, the use of linker moieties that cannot penetrate cellular membranes, such as bulky carbohydrates of charged linkers — bresidualizing labelsQ [12], should improve the intracellular retention of radiohalogens, including radiobromine. Our strategy was to develop a number of potentially residualizing radiobromine labels and select the best ones after biological characterization in vitro and in vivo. So far, our efforts have mainly been concentrated on developing negatively charged linkers on the basis of polyhedral boron anions, such as benzyl-isothiocyanate derivatives of closododecaborate [13] or nido-dicarborate [14]. Preliminary results of biological evaluation of such linkers are encouraging, especially in the case of closo-dodecaborate derivatives. However, it would be a significant mistake to neglect the potential of halogen linkers that are positively charged at lysosomal pH. Such linkers have been proposed by Garg et al. [15] and carefully evaluated for iodine and astatine radioisotopes by researchers at Duke University [15–29]. Related labeling chemistry has been elaborated by these researchers, and the results of thorough biological evaluation have demonstrated the appreciably improved targeting of radioiodine and astatine with the use of such linkers. We may expect that similar improvement of targeting could be achieved in the case of radiobromine as well. N-Succinimidyl 5-bromo-3-pyridinecarboxylate has been selected as a first candidate for our studies.

Given that bromine is more difficult to oxidize than is iodine or astatine, we selected Chloramine-T, a more potent oxidant than N-chlorosuccinimide as used by the Duke University researchers. Our previous experience [13,14] suggests that the use of an aqueous solution for precursor labeling and a bone-potQ labeling approach, excluding intermediate purifications, can considerably simplify the labeling procedure and improve yields. This prompted us to reevaluate labeling chemistry. We plan to use radionuclide targeting to the HER2 (neu, c-ErbB2) receptor. HER2 is a transmembrane protein belonging to the human epidermal growth factor tyrosine kinase receptor family. Increased HER2 activity is associated with increased proliferation and decreased apoptotic capacity. It is known that HER2 is not or only slightly expressed in normal adult tissues [30,31]. On the other hand, many breast, ovarian and urinary bladder carcinomas demonstrate great expression of HER2 [32,33], and this expression is believed to be a part of formation of the malignant phenotype [34]. Importantly, it was demonstrated that expression of HER2 has prognostic and predictive value in breast cancer [35–37]. Overexpression of HER2 is associated with short disease-free and overall survival. Overexpression of HER2 is also a marker of lack of efficacy of adjuvant tamoxifen therapy of breast cancer, especially in node-positive patients [35,38]. In contrast, tumors with HER2 overexpression are more sensitive to anthracycline (e.g., doxorubicin)-based chemotherapy [39]. For this reason, visualization of HER2 in tumors may improve patient management. In this study, commercially available mAb trastuzumab was selected for targeting HER2 on tumors. Trastuzumab is a humanized anti-HER2 antibody, which in nonlabeled form is used for the therapy of breast cancer [40,41]. Low rate of human antimouse antibody response associated with trastuzumab enables its repeated use, which is important for diagnostics. Trastuzumab is an internalizing mAb, and the effect of the residualizing label should be helpful in this case. The goal of the study was to evaluate the chemistry of trastuzumab radiobromination using N-succinimidyl 5-bromo-3-pyridinecarboxylate (2), with a view to its future use in immunoPET.

2. Methods 2.1. Materials Organic solvents were purchased from Merck (Darmstadt, Germany), Chloramine-T (CAT) and sodium metabisulfite from Sigma (St. Louis, MO, USA); all these chemicals were of analytical grade or better. Borate buffer was prepared using NaB4O710H2O (Sigma) and HCl (Merck). Phosphate buffered saline (PBS, 5 mM, pH 7.4) was prepared from Na2HPO410H2O, NaH2PO4H2O and NaCl, all of analytical grade (Merck). 5-Bromo-nicotinic acid and di-(N-succinimidyl) carbonate were obtained from

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2.3. Radiobromine production and precursor synthesis

Aldrich (St. Louis, MO, USA). Anti-HER2 mAb trastuzumab (Genentech) was purchased commercially and was supplied in pharmaceutical form. For labeling, the trastuzumab was purified from low-molecular-weight compounds by size-exclusion chromatography on a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with deionized water and then freeze-dried. Highquality Milli-Q water (resistance higher than 18 MV/cm) was used for preparing solutions and buffers. Solutions of CAT, sodium metabisulfite and N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate (1) were prepared immediately before use. For column chromatography, Merck silica gel 60 (230–400 mesh) and a mixture of CH3CN/CH2Cl2 (70:40) were used. Thin layer chromatography (TLC) was performed using Merck Silica 60 F254 gel plates and a neat acetonitrile as a solvent.

A mixture of bromine radioisotopes was used for the radiobromination study. These nuclides were produced by irradiating copper (II) selenide of natural isotopic composition with 40-MeV protons. Radioactivity measurements of the irradiated targets and produced radiobromine were carried out using gamma spectroscopy, as described in the Instrumentation section. Proton bombardment induces the bromine isotopes 77Br, 76Br and 82 Br, as well as various radioisotopes of copper, zinc, selenium and arsenic, in the target material. Bromine was separated from the target by dry distillation coupled with on-line thermochromatography purification [3] and was obtained as a no-carrier-added bromide solution in 200– 400 Al of ultra-pure water. Typical production batch contained 290 MBq 77Br, 228 MBq 76Br and 6 MBq 82 Br at the end of separation. No radioactive nuclides other than bromine isotopes were found in the product. Measurements of specific radioactivity were not performed for this preparation. Earlier, we produced 76Br using the same technique. The specific activity of the product was about 40 times lower than the theoretical value, 200 GBq/ Amol [43]. Presence of fairly long-lived 77Br (T 1/2 = 57 h) enabled the use of the radioactivity of a single batch for up to several days after radionuclide production. The mixture of bromine isotopes is designated from now on as *Br. Synthesis of N-succinimidyl 5-(tri-N-butylstanyl)-3pyridinecarboxylate (1) and N-succinimidyl 5-bromo-nicotinic acid (4) is outlined in Scheme 1. N-Succinimidyl 5-(tri-N-butylstanyl)-3-pyridinecarboxylate (1) was prepared according to the procedure described by Garg et al. [15]. The compound was characterized by NMR, and the NMR spectra data were found to be consistent with those reported in the literature. N-Succinimidyl 5-bromo-nicotinic acid (4) was prepared as follows. To a round-bottomed flask containing 100 ml acetonitrile were added 5-bromo-nicotinic acid (1.04 g, 5.15 mmol), pyridine (0.5 m1, 6.18 mmol) and di-(Nsuccinimidyl) carbonate (1.58 g, 6.18 mmol). The mixture

2.2. Instrumentation Radioactivity measurements were carried out with an ultra-pure germanium detector (ORTEC, Oak Ridge, TN, USA) connected to an 8192-channel PC-based multichannel analyzer (The Nucleus, Oak Ridge, TN, USA). The detector was calibrated for energy and efficiency with a standard 152 Eu source. Dead-time losses were always below 10% during measurements. Gamma-ray energies and abundance were taken from the Table of Radioactive Isotopes [42]. Alternatively, the radioactivity was measured with the automatic 1480 WIZARD 3-in. Gamma Counter (Wallac) equipped with a 3-in. NaI(Tl) well detector. The 1H and 13C spectra were recorded in CDCl3 (7.26 ppm 1H, 77.0 ppm 13 C) on a Varian Unity 400 spectrometer operating at 400 and 100.6 MHz, respectively, or a Varian Unity 500 spectrometer operating at 500 and 125 MHz, respectively. Distribution of radioactivity along the TLC strips was measured on the Cyclone Storage Phosphor System and analyzed using the OptiQuant image analysis software. Sizeexclusion chromatography was performed on disposable NAP-5 columns (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. In the cell-binding study, cells were counted using a cell counter (Z2, Beckman Coulter, Fullerton, CA, USA).

O

O

O

Br

I

OH

615

Br O

N

N

N

O

Ref. Garg S., et al, 1991

O

O

O

O

O N

N O

O *Br

*Br

(Bu)3Sn

O

II

N

N

MAb

N

O

Scheme 1. Reagents and conditions: (I) di(N-succinimidyl) carbonate, pyridine, MeCN, N2, 608C; (II) NH2, pH 9.3.

N H

III

76

Br , Chloramine-T, MeOH/HOAc, RT; (III) mAb-

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was stirred at 608C for 4 h, and the solvent was evaporated to dryness. The crude product was purified by flash chromatography using CH3CN/CH2Cl2 (70:40) as a mobile phase to give compound 4 as a white solid (1.06 g, 69% yield). 1 H NMR spectral data were in accord with those published previously [15].13C NMR (CDCl3): d 168.8, 160.0, 156.6, 149.5, 140.3, 123.2 121.1, 25.9. 2.4. Radiobromination of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate (1) The precursor molecule (1) was radiobrominated using CAT as oxidant (Scheme 1). Labeling yield dependence on the amount of added CAT and Nsuccinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate as well as on the reaction time was determined by varying one parameter at a time. In an Eppendorf tube (1.5 ml), 5 Al of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate solution in 5% acetic acid in methanol was mixed with 3 Al of *Br in Milli-Q water (containing 1–3 MBq 77 Br) and 10 Al of 0.1% acetic acid in water. To this mixture, 20 Al of CAT solution in water was added. The N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate amount varied between 1.9 and 76 nmol, and the CAT amount varied between 0 and 80 Ag (0.28 Amol). The Eppendorf tube was shaken lightly throughout the reaction time, which ranged from 1 to 10 min. The reaction was terminated by adding 20 Al of sodium metabisulphite in aqueous solution given in double molar excess to CAT. All experiments were made at least in duplicate. 2.4.1. Varying CAT amount When the amount of CAT was varied, the amount of added N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate remained constant at 10 Ag, and the reaction time was 2 min. 2.4.2. Varying N-succinimidyl 5-(tributylstannyl)-3pyridinecarboxylate amount When the amount of N-succinimidyl 5-(tributylstannyl)3-pyridinecarboxylate was varied the amount of added CAT remained constant at 20 Ag, and the reaction time was 1 min. Blank experiments were performed, when 5% acetic acid in methanol was used instead of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate solution. 2.4.3. Varying reaction time When the reaction time was varied the amount of added N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate remained constant at 10 Ag, and the amount of added CAT was 80 Ag. Silica gel 60 F254 TLC plates (20100 mm, elution path 80 mm; E. Merck) were used for analysis. Neat acetonitrile was used for elution. For the analysis, 1–2 Al of the reaction mixture was applied on a TLC plate, which was then left to evaporate spontaneously before being developed in the freshly prepared eluent.

2.5. Conjugation of [*Br] N-succinimidyl 5-bromo-3pyridinecarboxylate to trastuzumab (see Scheme 1) When [*Br] N-succinimidyl 5-bromo-3-pyridinecarboxylate was prepared for further conjugation to the mAb, radiobromide solution (100 Al containing approximately 60 MBq 77Br) was evaporated to dryness at 1008C under a gentle flow of argon gas, and the rest was redissolved in 10 Al of 0.1% acetic acid in water. N-Succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate (5 Ag in 2 Al of 5% acetic acid in methanol) and 10 Ag of CAT in 20 Al of water were added and mixed for 2 min. Sodium metabisulfite (20 mg in 10 ml of water) was added, and the mixture was vortexed for a few seconds. A small sample (0.5 Al) of the reaction mixture was taken for radio-TLC analysis. Trastuzumab (100 or 300 Ag, 10 mg/ml in borate buffer, pH 9.3) was added, and the mixture was incubated at room temperature. The reaction molar ratio was 5 and 15:1 for the active ester and the antibody, respectively. At predetermined time points, aliquots were taken, and the labeling yield was determined by size-exclusion chromatography as the ratio of radioactivity in the high-molecularweight (HMW) fraction to the sum of radioactivities in the HMW fraction, in the low-molecular-weight (LMW) fraction and on the column. All experiments were performed in duplicate. To verify that the labeling was associated with N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate, blank experiments were performed in duplicate. In this case, 5% acetic acid in methanol was used instead of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate solution, but all the manipulations were the same as in the case of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate labeling. 2.6. Stability test The in vitro stability of radiobromine binding to trastuzumab was assessed by using 8 M urea to achieve high ion strength, 1 M NaBr to challenge with an excess of nonradioactive bromide and calf serum at 378C to mimic physiological conditions. The labeling was performed according to the above method. To Eppendorf tubes (1.5 ml) containing 1 ml of each of the stability test solutions, 100 Al of radiobrominated trastuzumab was added. The tubes were shaken vigorously for 15 s. The NaBr and urea solutions were kept at room temperature for 1.5 h of light agitation, and fractions of 100 Al were taken for analysis. The calf serum sample was kept at 378C for 72 h, and 100-Al samples were taken for analysis. The samples were analyzed using size-exclusion chromatography, and the portion of radioactivity in the LMW fraction was assumed to have resulted from release of the label. A freshly radiobrominated solution of trastuzumab was analyzed in the same way to verify resolution of the system. All tests were performed in triplicate.

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2.7. Cell culture Human ovarian cancer cell line, SKOV-3 (American Type Culture Collection, Rockville, MD, USA), was cultured in McCoy’s 5a medium supplemented with 10% fetal calf serum, 2 mM l-glutamine and PEST (100 IU/ml penicillin, 100 Ag/ml streptomycin), all obtained from Biochrom (Berlin, Germany). Cells were cultured in Petri dishes (F 3.5 cm) at 378C in a humidified incubator containing 5% CO2. Cell dishes were washed with new medium, and three dishes were treated with trypsin-EDTA solution (0.05% trypsin, 0.02% EDTA in buffer, Flow Irvine, UK) for cell counting before the start of every experiment. 2.8. Immunoreactive fraction Labeling of trastuzumab for this experiment provided about two pendant groups per antibody molecule. Cells were placed into six Eppendorf tubes (5 million cells per tube). For estimation of nonspecific binding, HER2 receptors on cells were blocked in three tubes by adding 100 Ag of nonlabeled trastuzumab. Thereafter, 12.4 ng of radiolabeled antibody was added in each Eppendorf tube, and the tubes were vigorously vortexed. Incubation proceeded under slight shaking for 5 h at 48C. Cells were pelleted by centrifugation during 5 min at 9000g, and the supernatant was withdrawn. Radioactivity of cell pellet and supernatant was measured by automatic gamma counter, and percent of cell-associated radioactivity was determined for nonblocked and blocked cells. Immunoreactive fraction was determined as percent of cell-bound radioactivity in nonblocked cells minus percent of cell-bound radioactivity in blocked cells. 2.9. Comparative cellular retention of trastuzumab labeled using N-succinimidyl 5-bromo-3-pyridinecarboxylate and N-succinimidyl 4-bromobenzoate in SKOV-3 cells Cellular retention of trastuzumab radiobrominated using N-succinimidyl 5-bromo-3-pyridinecarboxylate was compared with retention of trastuzumab radiobrominated using N-succinimidyl 4-bromobenzoate. Radiobromination of trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate for this experiment provided about two pendant groups per antibody molecule. Bromobenzoate-labeled trastuzumab was prepared according to the procedure described by Hfglund et al. [10]. An average conjugate contained about 2.6 pendant groups per antibody. For each conjugate, a binding specificity test was performed before cellular retention study, which confirmed that cell binding was receptor-specific, since it could be displaced with large excess of nonlabeled trastuzumab (data not shown). To evaluate cellular retention of radioactivity after interrupted incubation, 0.3 Ag of each conjugate was added to cell dishes, containing about 105 cells each. Cells were incubated with conjugate for 2 h at 378C, and incubation media were removed from cells. Non-cell-bound antibody was removed from the cells by six times washing with cold

617

serum-free media. One milliliter of fresh complete media was added in each dish, and cells were incubated during 24 h at 378C. Thereafter, incubation media were collected, cells were detached from dishes by treatment with trypsin-EDTA solution and radioactivity of media and cells was measured. The fraction of the initially cell-associated radioactivity, which was still associated with cells, was determined as an average value for three samples.

3. Results 3.1. Radiobromination of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate Analysis of the reaction mixture after radiobromination typically revealed three radioactive products having the same R f as do nonradioactive standards of bromonicotinic acid (peak 1, R f = 0.1), bromide (peak 2, R f = 0.35) and [*Br] N-succinimidyl 5-bromo-3-pyridinecarboxylate (peak 3, R f =0.8). Peak 2 had the same R f as did the peak of radioactive bromide, and it was the only peak that appeared in radiochromatograms in blank experiments, i.e., experiments that included all manipulations performed for labeling, but with the addition of neat solvents instead of a solution of CAT or N-succinimidyl 5-(tributylstannyl)-3pyridinecarboxylate. On the contrary, peaks 1 and 3 never appeared in blank experiments, and their formation was associated with the treatment of radiobromide with an oxidant in the presence of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate (Fig. 1). All this gives us good reason to assume that peak 1 represents [*Br] bromonicotinic acid, peak 2 radiobromide and peak 3 [*Br] N-succinimidyl 5-bromo-3-pyridinecarboxylate. Dependence of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate radiobromination yield on reaction time was investigated. Radiobromination appeared to be rapid, and the maximum yield, 67F2%, was achieved within 1 min. With this amount of oxidant, which enabled secure oxidation of radiobromide, essentially all the bromide was consumed, and the only byproduct was [*Br] bromonicotinic acid. Increasing the reaction time beyond 1 min did not increase the formation of [*Br] N-succinimidyl 5-bromo-3pyridinecarboxylate, but made labeling less reproducible due to poorly controlled hydrolysis of the product and the formation of [*Br] bromonicotinic acid. Dependence of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate bromination yield on amount of substrate is shown in Fig. 2. The plateau value, 68.5F0.8%, was achieved in this series of experiments at a substrate amount of 5 Ag (9.5 nmol). Above this amount of substrate, a major product, besides [*Br] N-succinimidyl 5-bromo-3-pyridinecarboxylate, was [*Br] bromonicotinic acid. Nonreacted bromide always accounted for less than 5% of total radioactivity. When less substrate was taken (1 Ag, 1.9 nmol), the labeling yield decreased to 25.3F2.3%, mainly due to the increased amount of free unreacted radiobromide.

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A

B 20719

26355

1

13177

10359

5179

6588

0 0.0

2

15539

3 Activity(DLU)

Activity(DLU)

19766

O

25.0

50.0

75.0

0 0.0

100.0

F

Distance(mm)

O

25.0

50.0

75.0

Distance(mm)

F

100.0

Fig. 1. Typical radio-TLC chromatogram of the reaction mixture system: silica-coated plate, acetonitril as eluent. (A) Peak 1 (R f 0.1), [*Br] bromonicotinic acid; peak 3 (R f 0.8), [*Br] N-succinimidyl 5-bromo-3-pyridinecarboxylate. (B) Peak 2 (R f 0.35), bromide.

Influence of the amount of CAT on labeling yield is depicted in Fig. 3 (note logarithmic scale on the x axis). Plateau (69.8F1.6%) in this dependence was reached when 2 Ag of CAT was used. The use of 1 Ag led to noticeable decrease of the yield, down to 52.4F1.3%, accompanied by an increased fraction of free radiobromide. No radiobromodestannylation occurred when the CAT amount was 0.4 Ag or less, and all radioactivity was in the bromide chemical form. 3.2. Coupling of [*Br] N-succinimidyl 5-bromo-3-pyridinecarboxylate to trastuzumab In this experiment, two samples of trastuzumab (500 Ag each) were labeled according to standard protocol. Overall labeling efficiency, measured as ratio of radioactivity in HMW fraction after size-exclusion chromatography to the sum of radioactivity in the HMW and LMW fractions and on the column, was in this case more than 60%. To insure that the radiobromination of trastuzumab is really mediated

by [*Br] N-succinimidyl 5-bromo-3-pyridinecarboxylate, a blank experiment was performed. Two samples were handled in the same way, but a solution of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate was replaced with a neat solvent. No radioactivity was observed in the HMW fraction, which demonstrated that coupling of radiobromine to trastuzumab was associated with the use of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate. The time course of the coupling of [*Br] N-succinimidyl 5-bromo-3-pyridinecarboxylate to trastuzumab in 0.07 M borate buffer, pH 9.3, at room temperature and with two different concentrations of mAb is shown in Fig. 4. The coupling was rapid, the plateau being reached within the first 10 min of incubation, and extending coupling time did not increase overall yield. The coupling had a pronounced dependence on the concentration of trastuzumab. Based on the data concerning radiobromination of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate in these reactions

Influence of substrate amount on labeling yield 80

Influence of amount of CAT on labeling yield 60

Labeling yield, %

Labeling yield, %

80

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0 0

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Fig. 2. Dependence of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate bromination yield on amount of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate. Other conditions were as follows: CAT, 20 Ag; reaction time, 1 min. Total volume in each experiment was 58 Al. Tests were performed using 1–3 MBq 77Br.

Fig. 3. Dependence of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate bromination yield on amount of CAT. Other conditions were as follows: N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate, 10 Ag; reaction time 2 min. Experiments were performed using 1–3 MBq 77Br.

E. Mume et al. / Nuclear Medicine and Biology 32 (2005) 613 – 622 100

Influence of coupling time on overall labelling Cell-associated radioactivity, % of added

yield at different amount of antibody 50

40

Yield, %

619

30

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0

80

60

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Incubation time, min 0.3 mg

0.1 mg

Fig. 4. Coupling of [*Br] N-succinimidyl 5-bromo-3-piridinecarboxylate to trastuzumab in 0.07 M borate buffer, pH 9.2, at room temperature. Data are presented as mean values of the results of two experimentsFmaximum errors. Other conditions were as follows: amount of substrate, 9.5 nmol; labeling yield of the first step, 67%; overall volume, 88 Al.

(68%), coupling efficiency was about 67% in the case of 300 Ag of antibody and about 30% in the case of 100 Ag. 3.3. Stability of radiobromine label on trastuzumab The stability of the radioactive label was verified by incubating the labeled mAb in various solutions. Results of the stability test are presented in Table 1. Stability was evaluated by comparing the amount of radioactivity in the LMW fraction after gel filtration of the protein solutions with the total radioactivity. The labeled antibody was stable in high ionic strength solutions, as well as during challenge with a large amount of nonradioactive bromide for the testing period (i.e., 1.5 h). Under physiological conditions, imitated by incubation in calf serum at 378C, the label was stable for at least 72 h, corresponding to more than four half-lives of the label. 3.4. Immunoreactive fraction The data from immunoreactive fraction determination test are given in Fig. 5. When no blocking antibody was Table 1 Stability test of labeled mAba Incubation solution

Incubation temperature

Fresh solution in PBS Calf blood serum 8 M urea 1 M NaBr

208C 378C 208C, light shaking 208C, light shaking

Incubation time (h)

Fig. 5. Immunoreactive fraction determination. The test was performed on suspension of ovarian cancer cell line SKOV-3 (5 million cells per test tube). Radiolabeled antibody (12.4 ng, approximately 32 000 cpm) was added in each test tube. In the blocked cells, binding sites were presaturated by adding 120 Ag of nonradioactive mAb. Cells were incubated with labeled trastuzumab for 5 h at 48C, and radioactivity in cells and supernatant was determined. Data are presented as mean values from three samples and maximum errors.

added, 82.0F7.4% of the radioactivity was associated with HER2-expressing cells. Addition of large excess of trastuzumab reduced cell-associated radioactivity to 5.1F 2.6% ( P b.0001). These results indicate a receptormediated binding, since the binding could be displaced with nonlabeled antibody. Results of this experiment show that immunoreactive fraction of trastuzumab after labeling is at least 77% (difference between overall and nondisplaced binding). 3.5. Cellular retention of radioactivity after interrupted incubation with radiobrominated trastuzumab Experiments on cellular retention in vitro did not reveal any difference between trastuzumab labeled using N-succinimidyl 5-bromo-3-pyridinecarboxylate and N-succinimidyl 4-bromobenzoate. Twenty-four hours after interrupted incubation with radiolabeled antibodies, cell-associated radioactivity was 33.8F1.5% for 5bromo-3-pyridinecarboxylate linker and 33.4F1.7% for 4-bromobenzoate.

Radioactivity in LMW fraction (%)

4. Discussion

0

2.91F0.96

24 72 1.5 1.5

4.75F0.30 4.52F2.66 8.59F1.30 0.62F0.25

The fact of the lower tumor localization of radioiodine than of radiometal labels on antibodies was noted in the 1980s (see, e.g., Khaw et al. [44]). Later, it was proved that this phenomenon could be explained by differences in the physicochemical properties of radiocatabolites after the internalization and cellular degradation of antibodies. The major radiocatabolite of directly radioiodinated mAbs, monoiodotyrosine, is lipophilic enough to diffuse through lysosomal and cellular membranes, while the radiocatabo-

a Radioactivity content in the LMW fraction after incubation in various media was considered as a measure of radiobromine fraction, which was released from labeled antibody during stability test. Three test solution samples were measured.

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E. Mume et al. / Nuclear Medicine and Biology 32 (2005) 613 – 622

lites of metal-labeled mAbs are not (for a more detailed analysis of this question, see our review [11]). The use of nondegradable and hydrophilic carbohydrate-based linkers for attaching radioiodine — the so-called residualizing labels — lets us substantially improve the cellular retention of internalizing proteins [12,45 – 47]. Still, the labeling chemistry of carbohydrate-based halogen labels is complicated, which has restricted their use. Thus, Stein et al. [48] stated that the delivery of absorbed dose using [131I]dilactitol-tyramine was limited by the low conjugation efficiency of prelabeled linker to mAbs. Low conjugation yields, of 30 –40%, and a possible aggregation of antibodies when using tyramine-cellobiose were observed [49]. An alternative approach to the design of residualizing labels, namely, the use of templates that are positively charged at lysosomal pH, has been proposed by the Duke University group. Reist et al. [19] demonstrated that the use of N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC), initially proposed as a linker stable to deiodinating enzymes [15], improves the intracellular retention of radioactivity in vitro and improves tumor-to-normal-tissue ratios in comparison with IodoGen labeling. Later, the same group widened this concept by introducing new positively charged linkers for radioiodine and astatine, such as N-succinimidyl 4-guanidinomethyl-3-halobenzoate [27– 29], or d-amino acid peptide containing lysine and tyrosine [24]. Since this concept worked well for heavier halogens, we found it logical to apply it also to radiobromination. However, the electronegativity of bromine forced us to replace the oxidant with one that is more potent, i.e., Chloramine-T. The general labeling strategy was also reconsidered. The original labeling method used a large amount of substrate (about 0.5 mg), intermediate purification of radiolabeled N-succinimidyl 5-halo-3-pyridinecarboxylate by chromatography and a change of solvent before coupling to mAb. Although this strategy may work well in the university laboratory with well-trained personnel, in our experience such a strategy has some weaknesses. First, simple transfer of a small amount of radioactive solution from vial to vial usually leads to a loss of at least 5% of the radioactivity. Similarly, part of the radioactive substance usually adheres to the vial walls and is not resolubilized during solvent change. Multiple manipulations increase the probability of human error. All these factors negatively affect the overall yield, creating problems when transferring the labeling technology from university laboratory to nuclear medicine department. To avoid such problems, we applied bone-potQ indirect protein and peptide labeling without intermediate purification whenever possible. Preconditions for applying such a technique are as follows: –

the use of aqueous media throughout the labeling process, while the use of lipophilic solvents is avoided or minimized,

– –

the use of low-molarity buffers in initial steps and high-molarity buffer in later ones, minimization of the total amount of precursor in order to minimize the modification of protein to be labeled.

We have successfully applied this strategy in a number of studies [13,14,50,51]. If a rather lipophilic nonlabeled precursor, such as N-succinimidyl 4-(trimethylstannyl) benzoate, is used, it may first need to be dissolved in methanol; small amounts (2–5 Al) of this solution are then added to the reaction mixture. We have found that the use of such small amounts of methanol does not negatively affect the immunoreactive fraction or binding affinity of antibodies labeled in this way. The same approach, i.e., predissolution in acidified methanol, was applied in this work, since the water solubility of N-succinimidyl 5-(tributylstannyl)-3pyridinecarboxylate was insufficient. Radiobromination of N-succinimidyl 5-(tributylstannyl)3-pyridinecarboxylate in aqueous media using ChloramineT was quick and efficient, essentially all the radiobromine being consumed within the first minute; in this respect, the process resembled the radiobromination of N-succinimidyl 4-(trimethylstannyl) benzoate [10]. However, a substantial difference from the results of the above-cited study was found in the appreciably higher hydrolysis of labeled conjugate, when more than 20% of the radioactivity existed in the form of bromonicotinic acid. Earlier, hydrolysis has been observed in the case of iodination and astatination using N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate [15,21]. The fact that N-succinimidyl esters of halopyridinecarboxylates tend to be more susceptible to hydrolysis than are esters of halobenzoates has been explained by heteroatom influence [21]. A possible advantage of using Chloramine-T over using tert-butyl hydroperoxide or N-chlorosuccinimide is that labeling does not require heating to 60 –658C [15], which simplifies the process. Efficient use of Chloramine-T requires slightly acidic media. This was achieved by using 0.1% aqueous acetic acid as a solvent for radiobromination; this provided a pH of about 5, and it was easy to overbuffer it during the next coupling step, which required basic pH values for deprotonating amino groups on the antibody lysines. Exclusion of intermediate purification simplifies and shortens the labeling process. On the other hand, nonseparated and nonreacted N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate, as well as pseudo-carrier N-succinimidyl 5-chloro-3-pyridinecarboxylate [15], remains in the reaction mixture and might compete with radioactive N-succinimidyl 5-bromo-3-pyridinecarboxylate for binding sites on mAb. Attachment of these moieties to an antibody may cause modification of its properties, and the potential loss of its immunoreactivity. For this reason, the total amount of substrate should be minimized. This study demonstrated that as little as 9 nmol of N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate could be efficiently radiobrominated. Taking into account coupling

E. Mume et al. / Nuclear Medicine and Biology 32 (2005) 613 – 622

efficiency, we could suppose that labeling 1 mg of mAb would provide attachment of a single pendant group per antibody molecule. Such a degree of modification should be well tolerated by antibodies. The time course of the coupling of radioactive N-succinimidyl 5-bromo-3-pyridinecarboxylate to mAb differed noticeably from that of radioactive N-succinimidyl 4-bromobenzoate [10]. In both cases, coupling efficiency was proportional to concentration of mAb. However, in the case of N-succinimidyl 4-bromobenzoate, coupling yield increased with time for at least 1 h. In the case of N-succinimidyl 5-bromo-3-pyridinecarboxylate, the coupling yield did not increase after the first 10 min, which could be explained by the increased tendency of N-succinimidyl 5-halo-3-pyridinecarboxylates to hydrolysis. The results of immunoreactivity assessment demonstrated that immunoreactive fraction of radiolabeled antibody was more than 75%, which is comparable with immunoreactive fraction reported for other antibodies and fragments radiohalogenated using N-succinimidyl 5-tri-n-butylstannyl 3-pyridinecarboxylate (65–80% [22], 69–89% [20]). Retention of radiobromine by HER2-expressing SK-OV3 cells after interrupted incubation with trastuzumab labeled using N-succinimidyl 5-bromo-3-pyridinecarboxylate was evaluated and compared with retention of this antibody labeled using N-succinimidyl 4-bromobenzoate. Disappointingly, the results of this assessment did not demonstrate the advantage of the use of the linker that is charged at lysosomal pH. These results contradict literature data obtained for other radiohalogens (see, e.g., Ref. [17]). It should be noted that earlier results have been obtained for different antigen–antibody systems. The antigen-related differences in cellular processing of antigen–antibody complex may be a reason for the difference in cellular retention. Therefore, it might be possible that the use of N-succinimidyl 5-bromo-3-pyridinecarboxylate for labeling of other antibodies will improve their imaging properties. In conclusion, a method of indirect radiobromination of mAb trastuzumab using N-succinimidyl 5-bromo-3pyridinecarboxylate has been proposed. The method provides an overall labeling efficiency of 45.5F1.2%, similar to the efficiency of the indirect iodination and astatination of antibodies using N-succinimidyl 5-halo-3pyridinecarboxylates. At the same time, the method does not include heating or intermediate purification and thus takes less time. Radiolabeled trastuzumab preserved more than 75% immunoreactivity after radiolabeling. In vitro test on SKOV-3, however, did not show that the use of N-succinimidyl 5-bromo-3-pyridinecarboxylate instead of N-succinimidyl 4-bromobenzoate improves cellular retention of trastuzumab-delivered radiobromine. Acknowledgments This work was financially supported by grants from the Swedish Cancer Society (Cancerfonden). The authors thank

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Prof. Jfrgen Carlsson (Division of Biomedical Radiation Sciences, Uppsala University) for initiating research into tumor targeting using trastuzumab in our laboratories; his work has inspired this study among others. The authors also wish to thank the personnel of the Svedberg Laboratory, Uppsala, Sweden, and especially Mr. Lars Einarsson for technical support in radiobromine production. References [1] Lundqvist H, Lubberink M, Tolmachev V, Lovqvist A, Sundin A, Beshara S, et al. Positron emission tomography and radioimmunotargeting — general aspects. Acta Oncol 1999;38:335 – 41. [2] Lundqvist H, Tolmachev V. Targeting peptides and positron emission tomography. Biopolymers 2002;66:381 – 92. [3] Tolmachev V, Lfvqvist A, Einarsson L, Schultz J, Lundqvist H. Production of 76Br by a low-energy cyclotron. Appl Radiat Isot 1998; 49:1537 – 40. [4] Tolmachev V, Carlsson J, Lundqvist H. A limiting factor for the progress of radionuclide based diagnostics and therapy; availability of suitable radionuclides. Acta Oncol 2004;43:264 – 75. [5] Lovqvist A, Lundqvist H, Lubberink M, Tolmachev V, Carlsson J, Sundin A. Kinetics of 76Br-labeled anti-CEA antibodies in pigs; aspects of dosimetry and PET imaging properties. Med Phys 1999; 26:249 – 58. [6] Lubberink M. Quantitative imaging with PET: performance and applications of 76Br, 52Fe, 110mIn and 134 La. PhD Thesis, Uppsala University, Uppsala; 2001. [7] Lovqvist A, Sundin A, Ahlstrom H, Carlsson J, Lundqvist H. 76BrLabeled monoclonal anti-CEA antibodies for radioimmuno positron emission tomography. Nucl Med Biol 1995;22:125 – 31. [8] Lovqvist A, Sundin A, Ahlstrom H, Carlsson J, Lundqvist H. Pharmacokinetics and experimental PET imaging of a bromine-76labeled monoclonal anti-CEA antibody. J Nucl Med 1997;38:395 – 401. [9] Sundin J, Tolmachev V, Koziorowski J, Carlsson J, Lundqvist H, Welt S, et al. High-yield direct 76Br-bromination of monoclonal antibodies using the Chloramine-T. Nucl Med Biol 1999;26:923 – 9. [10] Hfglund J, Tolmachev V, Orlova A, Lundqvist H, Sundin A. Optimized indirect 76Br-bromination of antibodies using N-succinimidyl para-[76Br]bromobenzoate for radioimmuno PET. Nucl Med Biol 2000;27:837 – 43. [11] Tolmachev V, Orlova A, Lundqvist H. Approaches to improvement of cellular retention of radiohalogen labels delivered by internalizing tumor targeting proteins and peptides. Curr Med Chem 2003;10: 2447 – 60. [12] Thorpe SR, Baynes JW, Chroneos ZC. The design and application of residualizing labels for studies of protein catabolism. FASEB J 1993; 7:399 – 405. [13] Bruskin A, Sivaev I, Persson M, Lundqvist H, Sjfberg S, Tolmachev V. Radiobromination of monoclonal antibody using isothiocyanato derivative of closo-dodecaborate ([76Br]Bromo-DABI). Nucl Med Biol 2004;31:205 – 11. [14] Winberg KJ, Persson M, Malmstrfm PU, Sjfberg S, Tolmachev V. Radiobromination of anti-HER2/neu/erbB-2monoclonal antibody using the p-isothiocyanatobenzene derivative of the [76Br]undecahydro-bromo-7,8-dicarba-nido-undecaborate(1 ) ion. Nucl Med Biol 2004;31:425 – 33. [15] Garg S, Garg PK, Zalutsky MR. N-Succinimidyl 5-(trialkylstannyl)-3pyridinecarboxylates: a new class of reagents for protein radioiodination. Bioconjug Chem 1991;2:50 – 6. [16] Garg S, Garg PK, Zhao XG, Friedman HS, Bigner DD, Zalutsky MR. Radioiodination of a monoclonal antibody using N-succinimidyl 5-iodo-3-pyridinecarboxylate. Nucl Med Biol 1993;20:835 – 42. [17] Reist CJ, Garg PK, Alston KL, Bigner DD, Zalutsky MR. Radioiodination of internalizing monoclonal antibodies using

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