Development of a Streptavidinâ Anti-Carcinoembryonic Antigen

Bioconjugate Chem. 1997, 8, 585−594

585

Development of a Streptavidin-Anti-Carcinoembryonic Antigen Antibody, Radiolabeled Biotin Pretargeting Method for Radioimmunotherapy of Colorectal Cancer. Reagent Development† Habibe Karacay,*,‡ Robert M. Sharkey,§ Serengulam V. Govindan,‡ William J. McBride,‡ David M. Goldenberg,§ Hans J. Hansen,‡ and Gary L. Griffiths‡ Immunomedics, Inc., Morris Plains, New Jersey 07950, and Garden State Cancer Center, Belleville, New Jersey 07109. Received February 25, 1997X

With pretargeting, radioisotope delivery to tumor is decoupled from the long antibody localization process, and this can increase tumor:blood ratios dramatically. Several reagents were prepared for each step of a “two-step” pretargeting method, and their properties were investigated. For pretargeting tumor, streptavidin-monoclonal antibody (StAv-mab) conjugates were prepared by cross-linking sulfoSMCC-derivatized streptavidin to a free thiol (SH) group on MN-14 [a high-affinity anti-carcinoembryonic antigen (CEA) mab]. Thiolated mabs were generated either by reaction of 2-iminothiolane (2-IT) with mab lysine residues or by reduction of mab disulfide bonds with (2-mercaptoethyl)amine (MEA). Both procedures gave protein-protein conjugates isolated in relatively low yields (20-25%) after preparative size-exclusion (SE) chromatography purification with conservative peak collection. Both StAv-MN-14 conjugates retained their ability to bind to CEA, to an anti-idiotypic antibody to MN-14 (WI2), and to biotin, as demonstrated by SE-HPLC. Two clearing agents, WI2 mab and a biotin-human serum albumin (biotin-HSA) conjugate, were developed to remove excess circulating StAv-MN-14 conjugates in animals. Both clearing proteins were also modified with galactose residues, introduced using an activated thioimidate derivative, to produce clearing agents which would clear rapidly and clear primary mab rapidly. At least 14 galactose residues on WI2 were required to reduce blood levels to 5.9 ( 0.7% ID/g in 1 h. Faster blood clearance (0.7 ( 0.2% ID/g) was observed in 1 h using 44 galactose units per WI2. For the delivery of radioisotope to tumor, several biotinylated conjugates consisting of biotin, a linker, and a chelate were prepared. Conjugates showed good in vitro and in vivo stability when D-amino acid peptides were used as linkers. biotin-peptide-DOTAindium-111 had a slightly longer blood circulation time (0.09 ( 0.02% ID/g in 1 h) than biotin-peptideDTPA-indium-111 (0.05 ( 0.03% ID/g in 1 h) in nude mice. A longer circulation time with the neutral DOTA complex might allow higher tumor uptake.

INTRODUCTION

The concept of monoclonal antibody (mab)-directed pretargeting for the delivery of radioimmunotherapy (RAIT) to target cells in vivo has received considerable attention in the last few years. A significant advantage of using this approach is the fact that the lengthy antibody-to-target localization phase occurs with the mab not carrying a radiotoxic nuclide. Instead, the mab carries a secondary recognition moiety, which itself is later targeted with a radiolabeled hapten recognizing that secondary recognition moiety. A number of such systems have been described (1-5), with the best known being the one based on the high-affinity interaction between avidin (or streptavidin) and biotin (6-12). Basically, two biotin-avidin pretargeting approaches have been described. The “two-step” method involves administration of a mab-avidin (-streptavidin) conjugate followed by a biotin-nuclide conjugate. In the “three-step” method, mab-biotin is administered first, followed by an avidin (which acts as a mab-biotin clearing agent and biotin-nuclide bridging agent), and † Presented in part at the Sixth Conference on Radioimmunodetection and Radioimmunotherapy of Cancer in Princeton, NJ, on October 10-12, 1996. * Address correspondence to this author at Immunomedics, Inc., 300 American Rd., Morris Plains, NJ 07950. Telephone: (201) 605-8200. Fax: (201) 605-1103. ‡ Immunomedics, Inc. § Garden State Cancer Center. X Abstract published in Advance ACS Abstracts, July 1, 1997.

S1043-1802(97)00102-X CCC: $14.00

third a biotin-nuclide conjugate. In both methods, additionally administered agents which remove residual primary conjugate from the blood have been described (13, 14). Perhaps because of the highly interdisciplinary nature of this field, many publications only deal with the chemistry involved in reagent preparation in a peripheral manner. However, since the reagents necessitated by the approach include protein-protein conjugates, proteincarbohydrate conjugates, biotinylated proteins, radiolabeled proteins, biotin-chelate conjugates, and radiolabeled biotin-chelate conjugates, the required supporting chemistry is neither obvious nor trivial. Aside from synthetic chemistry and bioconjugate chemistry work, methods of purification and analysis also need to be developed for each prospective agent prior to embarking on expensive in vivo experimentation. A number of articles have appeared which outline various methodologies in this area, but these methodologies are often incompletely developed, sometimes appear less than satisfactory in terms of analytical characterization, and, in any case, are scattered across the scientific literature (15-28). In this paper, we concentrate on chemical considerations related to preclinical two-step pretargeting studies, and we identify and describe useful preparative and analytical methods for this purpose. The reagents described herein are for evaluation of two-step pretargeting, using appropriate radiolabeled derivatives, although more than two steps are used. © 1997 American Chemical Society

586 Bioconjugate Chem., Vol. 8, No. 4, 1997

Tumor is pretargeted with StAv-MN-14, a streptavidinanti-carcinoembryonic (CEA) mab conjugate, and after allowing 2 days for maximizing tumor uptake, a clearing agent is injected to remove non-tumor-bound, circulating StAv-MN-14. A clearing agent is used to remove extraneous conjugate from the blood and achieve high tumor:blood ratios of StAv-MN-14 in a short time. An alternative approach would be to allow a longer time for the conjugate to clear from blood without the aid of a clearing agent, as has been described (26). This approach requires that the StAv-mab conjugate remain at the tumor for a long period of time either without internalization or without losing its biotin binding capacity due to endogenous biotin-saturating StAv binding sites. Thus, a clearing agent is used to reduce the time between the targeting by the mab and the injection of the labeled biotin. Clearance mechanisms to be tested are based on anti-idiotypic binding to the antigen-binding part of StAv-MN-14 or biotin binding to the StAv part of the StAv-MN-14 conjugate. In addition, clearing agents substituted with carbohydrate moieties are examined, since these agents themselves are rapidly cleared from the blood and localized into hepatocytes by specific receptors (29). Radiometal-labeled biotinylated chelate is administered in the final step 2-24 h after the clearing agent. Requirements for a useful biotinylated chelate include in vivo stability, since serum biotinidase is known to cleave biotinylated substrates (30). A final requirement is the fact that the radiometal-chelate-biotin must have a reasonably short serum half-life in order to minimize normal tissue radiotoxicity upon therapy; yet at the same time, it must remain in circulation long enough to allow for adequate tumor accretion. MATERIALS AND METHODS

The murine anti-CEA antibody, MN-14, has been described previously (31), as has the rat anti-MN-14 idiotypic antibody WI2 (32). Streptavidin (StAv) and avidin were purchased from Pierce (Rockford, IL) and Sigma Chemical Co. (St. Louis, MO), respectively, and used without purification. Human serum albumin (HSA) (catalog no. A 3782) was from Sigma and was purified on a preparative size-exclusion HPLC column to remove a large molecular weight contaminant prior to its modification. CEA was obtained from Scripps (San Diego, CA). 1,4,7,10-Tetraazacyclododecane-N,N′,N′′,N”′-tetraacetic acid (DOTA) was obtained from Parish Chemicals (Orem, UT). (2-Mercaptoethyl)amine (MEA) and cyanomethyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside (CTTG) were from Sigma, and [5-(biotinamido)pentyl]amine was from Pierce. The remaining chemicals, including 2-iminothiolane, were purchased from Pierce or Aldrich (Milwaukee, WI). All were used without further purification. N,N,N′,N′′,N′′-Pentakis(carboxymethyl)-2-(p-aminobenzyl)diethylenetriamine (aminobenzyl-DTPA) was prepared according to literature methods (33) and converted to N,N,N′,N′′,N′′-pentakis(carboxymethyl)-2-(p-isothiocyanatobenzyl)diethylenetriamine (ITC-Bz-DTPA) as described (34). Protein solutions were sterile-filtered using Millipore (Marlborough, MA) Millex-GV 0.22 µm filter units. Centrifuged spin columns were prepared according to published procedures (34, 35). The gel, Sephadex G-5080 (Sigma), was hydrated in the appropriate buffer and washed with 7 buffer volumes before storage at 4 °C. Analytical protein analyses were performed on a Bio-Sil SEC-250 SE-HPLC column (Bio-Rad, Richmond, CA) equipped with an in-line absorbance detector (Waters 486 or Beckman 167), set at 280 nm, and an in-line radiation

Karacay et al.

detector (Packard Radiomatic Flo-One). Columns were eluted at 1 mL/min with 0.2 M sodium phosphate and 0.02% sodium azide at pH 6.8. Preparative protein purifications were carried out by fractionation on a Waters 650 HPLC system using a 60 × 21.5 cm TSK-gel G3000SW column (TosoHaas, Montgomeryville, PA), eluted with 0.2 M sodium phosphate, 0.15 M sodium chloride, and 0.02% sodium azide at pH 6.8 at 1.5 mL/ min. Concentrations of StAv-MN-14 were determined from a calibration curve (concentration vs absorbance) constructed using absorbance readings from equimolar solutions of StAv and MN-14 at 280 nm. The curve was constructed by mixing 10-45 µL of 1.25 × 10-5 M solutions of StAv and MN-14 in 0.1 M sodium phosphate at pH 7 to a final volume of 555 µL. The slope and intercept were determined to be 3.95 × 105 M-1 L and -0.0217, respectively. Biotin analyses were performed using a published spectrophotometric procedure (4′-hydroxyazobenzene-2carboxylic acid; HABA) (36, 37). The biotinylated peptides and chelates were analyzed on a Waters 4000 HPLC system using a reverse-phase Waters 8 × 100 mm radial pak cartridge filled with C-18 Nova-Pak 4 µm stationary phase where the column was eluted at 3 mL/min with a linear gradient of 100% A [0.1% trifluoroacetic acid (TFA) in water] to 100% B (0.1% TFA in 90% acetonitrile/10% water) over 10 min. At 10 min, the flow rate was increased to 5 mL/min and remained at 100% B for 5 min before the re-equilibration to initial conditions began. The purifications of the peptides and chelating agents were performed on preparative C-18 column, Waters PrepPack RCM Base, using gradients of the above eluants at 75 mL/min. A Waters 486 absorbance detector set at 220 nm was used for both analytical and preparative HPLC systems involving biotinylated peptides. Mass spectral analyses were performed by Mass Consortium Corp. (San Diego, CA) using electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry methods for small molecular weight compounds and antibodies, respectively. For in vitro stability studies, blood drawn from healthy volunteers was allowed to clot at room temperature, centrifuged, and sterile-filtered. Serum aliquots (250 and 500 µL) were stored frozen at 4 °C until needed. Approximately 45 min before use, serum vials were thawed and equilibriated at 37 °C under a humidified 5% CO2 atmosphere. Mouse serum was isolated from non-tumorbearing 5-8-week-old athymic nude mice (Taconic Farms, Germantown, NY). Mice were first anesthesized with sodium pentobarbital and bled by cardiac puncture. Blood was allowed to clot and serum collected after centrifugation. The GW-39 animal xenograft (human colonic carcinoma) model is described in detail elsewhere (38). All experiments involving animals were carried out using AAALAC-recognized standard procedures for the humane use of such animals. StAv-MN-14 with StAv Appended to mab Lysine Residues. Streptavidin (20 mg, 3.3 × 10-7 mol) in 1.9 mL of 0.1 M sodium borate (pH 8.3) containing 10 mM ethylenediaminetetraacetic acid (EDTA) was treated with 80 µL of an aqueous solution of sulfosuccinimidyl 4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC, 0.8 mg, 1.8 × 10-6 mol) and stirred at room temperature for 1 h. The modified streptavidin (StAvSMCC) was purified on 4 × 3 mL centrifuged spin columns in 0.1 M PBS and 10 mM EDTA at pH 6.4. To 6.6 mL of MN-14 (47.6 mg, 3.1 × 10-7 mol) in 0.1 M PBS at pH 7.4 was added 600 µL of 0.5 M borate buffer at pH 8.5. A fresh solution of 0.1 M 2-IT in H2O was prepared, and 16 µL of this (1.6 × 10-6 mol) was added to the MN-

Reagent Development for a Biotin Pretargeting Method

14 solution. The mixture was stirred at room temperature for 50 min and the thiolated mab was purified on 8 × 3 mL spin columns in 0.1 M PBS and, 10 mM EDTA at pH 6.4. This effluent was added to the purified StAvSMCC solution and stirred at room temperature for 1 h. After this time, N-ethylmaleimide (10 mg, 8 × 10-5 mol) dissolved in 50 µL of N,N-dimethylformamide (DMF) was added to the conjugation reaction mixture and incubated for 30 min at 37 °C to block residual mab thiol groups. The StAv-MN-14 conjugate was purified on a preparative size-exclusion HPLC column, as described above. StAv-MN-14 with StAv Appended to mab Cysteine Residues. Streptavidin (25 mg, 4.2 × 10-7 mol) in 2.5 mL of 0.1 M sodium phosphate at pH 7.3 was treated with 208 µL of 10 mM sulfo-SMCC in H2O (2.1 × 10-6 mol) and incubated at room temperature for 45 min. The modified StAv was purified on 4 × 3 mL spin columns in 0.1 M sodium phosphate at pH 7, and its concentration was determined by UV absorbance (1%E280 ) 32). MN-14 (60 mg, 3.9 × 10-7 mol in 3 mL of 0.1 M sodium phosphate at pH 7.3 was treated with 600 µL of 0.1 M MEA in 5 mM EDTA. The reduction was carried out for 45 min at room temperature. Reduced MN-14 was purified on two successive sets of 5 × 3 mL spin columns with 0.1 M sodium phosphate and 5 mM EDTA at pH 7, and its final concentration was determined by UV absorbance at 280 nm. The number of free SH groups per mab was determined by Ellman reaction by reference to a standard curve (39). The StAv-MN-14 conjugate was prepared by simultaneous addition of 3.35 mL of MN-14 (IgG-SH) (3.05 × 10-7 mol) and 3.35 mL of StAv-SMCC (3.05 × 10-7 mol) in 5 equal portions over 10 min to 6 mL of 0.1 M sodium phosphate at pH 7.0 while stirring. After 1 h at room temperature under an argon atmosphere, solid sodium tetrathionate was added to a final concentration of 5 mM and the solution was stirred for an additional 5 min. The reaction mixture was first purified on 16 × 3 mL spin columns in 0.1 M sodium phosphate at pH 7.3 and then as above on a preparative SE-HPLC column. Retention times on analytical size-exclusion HPLC columns were as follows: streptavidin, 11.5 min; MN-14, 10.0 min; and StAv-MN14, 9.3 min. For radiolabeling purposes, both conjugates were substituted with the strong chelating agent, Bz-DTPA. Briefly, to a solution of StAv-MN-14 (325 µL, 4.6 mg, 2.15 × 10-8 mol) in 0.1 M sodium phosphate at pH 8.1 was added 4.1 µL of ITC-Bz-DTPA (1.4 × 10-7 mol). The pH of the reaction mixture was raised to 8.4 with 5-10 µL of a saturated solution of tribasic sodium phosphate, and the mixture was left at 37 °C for 90 min. The product was isolated following purification on two consecutive 3 mL spin columns in 0.1 M sodium acetate at pH 6.5. StAv-MN-14 Prepared Using Biotin. This conjugate was prepared according to a published procedure (40). MN-14 was first biotinylated by treating MN-14 (17 mg, 1.1 × 10-7 mol, 18.1 mg/mL) in 0.1 M sodium phosphate at pH 8.6 with 12 µL of 1 mg/100 µL solution of sulfo-NHS-LC-biotin (2.2 × 10-7 mol). After 2 h at 4 °C, the biotinylated mab was purified on two consecutive 3 mL spin columns packed in 0.1 M sodium phosphate at pH 7. HABA analysis demonstrated 0.8 biotin per MN-14. This mab (10.9 mg in 800 µL) was added to 1 mL of streptavidin (21 mg, 3.51 × 10-7 mol) in 0.1 M sodium phosphate at pH 7 in 5 × 200 µL portions. After a further 30 min, the StAv-biotin-MN-14 conjugate was purified by SE-HPLC, as described above. For each conjugate, retention of the biotin binding capacity was determined by mixing 10 µL aliquots of the

Bioconjugate Chem., Vol. 8, No. 4, 1997 587

StAv-MN-14 conjugate (1.2 × 10-7 M in 0.1 M sodium phosphate at pH 7.3) with 10 µL of 111In-bio-DTPA or bio-peptide-DOTA (0.3 µCi, 6 × 10-12 mol) (see below) in 0.1 M sodium acetate at pH 6.5 and determining the percentage of radioactivity associated with the conjugate by SE-HPLC. Values obtained were compared to those for free streptavidin (i.e., four biotin binding sites). Galactosylation of Proteins. These compounds were prepared using published methods (29, 41). Briefly, CTTG (0.23 g, 5.7 × 10-4 mol) was dissolved in 6.2 mL of anhydrous methanol. Sodium methoxide, 0.5 M in methanol (115 µL, 5.75 × 10-5 mol), was added to the above solution under argon and stirred at room temperature for 18 h. The imidate was used immediately, or unused portions were stored at 4 °C. Stored solutions were discarded upon the appearance of precipitate. To galactosylate lysine residues on WI2, imidate corresponding to various imidate:WI2 molar ratios was evaporated in individual vials using a stream of argon. WI2 mab was added to each, the final protein concentration adjusted to 8.4 mg/mL by addition of 0.1 M sodium phosphate at pH 8.1, and the final pH adjusted to 8.58.6 with a saturated solution of tribasic sodium phosphate. Reaction mixtures were stirred at room temperature for 2 h, and the modified WI2s were purified on two consecutive sets of centrifuged spin columns packed with Sephadex G-50-80 in 0.1 M sodium phosphate at pH 7.3. Initially, the level of galactose modification was determined indirectly by analyzing the degree of modification of protein primary amines using a published fluorometric assay (42). Galactosylated WI2 samples were later analyzed by MALDI-MS to determine the exact number of galactose residues present. HSA was biotinylated by treating 37.7 mg of purified HSA (5.54 × 10-7 mol) in 0.1 M sodium phosphate at pH 8.1 with 1.6 mg of sulfo-NHS-LC-biotin (2.88 × 10-6 mol) at a final protein concentration and pH of 15.6 mg/mL and 8.4, respectively. After 1 h at room temperature, the reaction mixture was applied to three sets of consecutive centrifuged spin columns to remove unreacted biotin. Biotin analysis by HABA showed 2.5 biotins per HSA. Several batches of biotinylated HSA were galactosylated with 100- and 300-fold molar excesses of dried imidate. Each reaction was performed at a final protein concentration of 10 mg/mL in 0.1 M sodium phosphate at pH 8.7, adjusted thereto with a saturated solution of tribasic sodium phosphate. After 2 h at room temperature with occasional vortexing, the conjugates were purified on two consecutive sets of spin columns with 0.1 M sodium phosphate at pH 7.5. [5-(Biotinamido)pentyl]amidyl-Bz-DTPA (Bio-DTPA). This reagent was prepared by incubating [5-(biotinamido)pentyl]amine (86.3 mg, 2.63 × 10-4 mol) and ITC-Bz-DTPA (6.39 × 10-5 mol) in 1.2 mL of sodium phosphate at 37 °C for 1 h while the pH was maintained at 9.2 by occasional addition of saturated tribasic sodium phosphate. RP-HPLC analysis indicated completion of the reaction by consumption of ITC-Bz-DTPA. The product was isolated in 68-83% yield following purification on a preparative C-18 column using a step gradient of 0% B for 10 min and 20% B for 9 min, followed by a linear gradient of 20 to 30% B over 30 min at a constant flow rate of 75 mL/min. The retention time on an analytical C-18 column was 6.29 min. The identity of the product was confirmed by ESI mass spectrometry [m/e 869 (M + H)+]. [(Biotinamido)pentyl]-DOTA Monoamide. DOTA was activated using a published procedure (43). Briefly, DOTA (81.5 mg, 0.2 mmol) was suspended in 10 mL of DMF/triethylamine (Et3N) (4:1 v/v) and stirred at room


temperature under argon for 18 h. The almost clear solution was cooled at 4 °C (ice bath) and was treated, under stirring, with a dropwise addition of isobutyl chloroformate (26.5 µL, 0.2 mmol) using an Eppendorf pipette. After 15 min, [5-(biotinamido)pentyl]amine (65.7 mg, 0.2 mmol) was added. The reaction mixture was stirred for 5 h at room temperature. The solvents were evaporated using a high-vacuum pump, and the residue was dissolved in 8 mL of water and injected onto a preparative reverse-phase HPLC column. The HPLC conditions were as follows: elution with 0% B for 10 min, followed by a linear gradient of 0 to 100% B over 60 min, with the solvent flow constant at 75 mL/min. Monobiotinylated DOTA (29 mg, 20.3% yield) was isolated as a white solid with a retention time of 5.57 min on the analytical RP-HPLC column; ESI mass spectrometry yielded a value of m/e 715 [M + H]. Biotin-D-Peptide-DTPAs. Peptides (1) biotin-D-LysNH2, (2) biotin-D-Phe-D-Lys-NH2, and (3) biotin-D-PheD-Phe-D-Lys-NH2 were synthesized either manually or on an automated peptide synthesizer (Advanced Chemtech 348 MPS) by the standard solid-phase methodology (44) on a Rink resin (Advanced ChemTech; 0.56 mmol/g) in which the N-R-amino groups were Fmoc-protected. After the last amino acid was attached, biotin was coupled using O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) (0.15 M), 1-hydroxybenzotriazole (HOBT) (0.15 M), and N,N-diisopropylethylamine (DIPEA) (0.75 M) in DMF. A minimal amount of Nmethylpyrrolidinone was added to aid in solubilization. The peptides were cleaved from the resin with a solution of 91% TFA/4.5% water/4.5% ethylmethyl sulfide. Subsequent to removal of the solvents, the crude peptides (1-3) were treated with a 0.3-0.5:1 molar ratio of ITCBz-DTPA/peptide in water at pH 8.6 and 37 °C until all of the ITC-Bz-DTPA was determined to be consumed by monitoring with RP-HPLC. The peptide bio-D-Phe-DPhe-D-Lys-NH2 required 20% acetonitrile for solubilization in water. The unreacted peptides and the biotinpeptide-DTPAs were isolated by preparative RP-HPLC purification using a linear gradient from 0 to 70% B over 40 min at 75 mL/min. Yield: peptides, 59-64%; peptide-DTPA, 68-76%. Mass spectrum m/z (M + H)+: peptide 1 not isolated; peptide 2 (C25H38N6O4S), calcd 519, found 519; peptide 3 (C34H46N7O5S), calcd 666, found 666; peptide-DTPA 1 (C38H57N9O13S2), calcd 912, found 912; peptide-DTPA 2 (C47H66N10O14S2), calcd 1059, found 1059; peptide-DTPA 3 (C56H75N11O15S2), calcd 1206, found 1206. Biotin-D-Peptide-DOTAs. DOTA was activated as its N-hydroxysuccinimide ester according to a published procedure (45). To a solution of 450 mg (960 µmol) of DOTA and 208 mg (960 µmol) of sulfo-NHS in 1 mL of water, cooled to 4 °C, was added 503 µL (91.5 mg, 477 µmol) of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), freshly prepared in water at 4 °C. The reaction mixture was stirred at 4 °C for 30 min, after which it was used immediately, without purification. Crude biotinyl-D-peptide (∼320 µmol of biotin-D-Lys-NH2, biotin-D-Phe-D-Lys-NH2, or biotin-D-Ser-D-Lys-NH2) dissolved in 500 µL of acetonitrile and 2 mL of water was added to the active ester and the pH of the reaction adjusted to 8.5 with 6 N NaOH. The mixture was stirred for 15 h at 4 °C and purified on a preparative RP-HPLC column with a gradient system of 0 to 15% B over 40 min at 75 mL/min. For biotinylated DOTA chelating agents, yields, retention times on an analytical RP-HPLC col-

Karacay et al.

umn, and ESI-MS data are as follows.

bio-D-Lys-(DOTA)-NH2 (C32H55N9O10S) bio-D-Phe-D-Lys-(DOTA)-NH2 (C41H64N10O11S) bio-D-Ser-D-Lys-(DOTA)-NH2 (C35H60N10O12S)

yield ret. time (mg) (min) 40 5.45

m/z (M + H)+ calcd found 758 758

87

6.3

905

905

50

5.63

845

845

Radiolabeling. MN-14, streptavidin, and StAv-MN14 were radioiodinated by the chloramine-T method (46). The StAv-MN-14-Bz-DTPA conjugate was radiolabeled with 88Y (Los Alamos National Laboratory, Los Alamos, NM). Briefly, 88YCl3 was neutralized with 3 volumes of 0.5 M sodium acetate at pH 6.1 and mixed with the StAv-MN-14-Bz-DTPA conjugate at a specific activity of 0.1 mCi/mg. After 1 h at room temperature, 0.1 M DTPA was added to a final concentration of 10 mM and the mixture was left for an additional 10 min before applying to two consecutive spin columns of Sephadex G-50-80 in 0.1 M sodium acetate at pH 6.5. Purified yields ranged from 70 to 90%. All radiolabeled conjugates were analyzed by SE-HPLC. The immunoreactive fraction of radiolabeled MN-14 and StAv-MN-14 was estimated from the percentage of radioactivity shifting to an earlier HPLC retention time after complexation with an 10-80-fold molar excess of CEA for 5 min at room temperature. Biotinylated chelating agents were radiolabeled with 111In. Biotinylated DTPAs (6.5 × 10-9 mol) in 122 µL of 0.5 M sodium acetate at pH 6.1 were added to 2.3 mCi 111InCl (received in 0.05 M HCl and was then diluted 3 with 3 volumes of 0.5 M sodium acetate at pH 6.1 before use) and incubated at room temperature for 1 h. The labeling yield was determined by analytical size-exclusion HPLC and by instant thin layer chromatography (ITLC) where approximately 0.2 µCi of the sample was applied to ITLC strips (Gelman Sciences) prespotted with 5 µL of 1% HSA and developed in a 5:2:1 water/ethanol/ ammonium hydroxide solvent system. In this ITLC system, unchelated 111In remains at the origin while the chelated 111In migrates with the solvent front. Radiolabeling of biotin-chelate was confirmed by HPLC analysis of the radiolabeled sample both before and after mixing with streptavidin, with successful radiolabeling indicated by a complete shift of retention time of the radioactive biotin-chelate (14-15 min) to the retention time of streptavidin (11.5 min). For biotinylated DOTA chelating agents, 2.4 mCi of InCl3 in 3 volumes of 0.5 M ammonium acetate at pH 5.5 was mixed with 21.5 µL of biotin-peptide-DOTA (4.7 × 10-8 mol) in 0.5 M ammonium acetate at pH 5.5. Mixtures were held for 2 h at 42-45 °C in a preheated block, and radiolabeling (g97%) was confirmed as for the DTPA-biotin-chelates. For in vitro stability studies, chelates were labeled with either 111In or 90Y as described and diluted in 250 µL of human serum to 1.2 × 10-7 to 2.1 × 10-6 M. Serum samples were incubated at 37 °C under a 5% CO2 atmosphere, and 5-10 µL aliquots were analyzed by HPLC before and after mixing with excess streptavidin (1-200-fold). The in vitro stability of biotinylated HSA was studied by incubating 20 µg of bio3-HSA-gal39 in 500 µL of mouse serum at 37 °C under 5% CO2. Samples (20 µL at 1 and 4 h) were mixed with 111In-Bz-DTPAStAv-MN-14 and analyzed by SE-HPLC. RESULTS

StAv-MN-14 Conjugates. StAv-MN-14 conjugate preparation using both methods (Figure 1) resulted in



Table 1. Biotin Binding Capacity of Streptavidin and StAv-MN-14 Conjugates protein

biotin binding capacity

streptavidin StAv-MN-14 (2-IT method) StAv-MN-14 (MEA method) StAv-biotin-MN-14

4 4 ( 0.3 4 ( 0.3 3 ( 0.5

Table 2. Determination of Galactose Levels on mab WI2 by MALDI-MS

Figure 1. StAv-MN-14 (IgG) was prepared by covalent attachment of activated streptavidin to thiolated IgG (IgG-2IT, method 1) or MEA-reduced IgG (IgG-SH, method 2).

Figure 2. (A) HPLC chromatograms of StAv-MN-14 (IgG) conjugates prepared by 2-IT (method 1) and MEA (method 2) on an analytical size-exclusion HPLC column. (B) Characterization of StAv-MN-14. Retention of its binding properties with gal-WI2 (1:1), bio3-HSA-gal39 (1:1), and 111In-bio plus CEA (20 × excess of CEA) as indicated by elution near the void volume on an analytical size-exclusion HPLC apparatus. Retention times: gal-WI2 (9.3 min), bio3-HSA-gal39 (11 min), and 111In-bio-DTPA (15 min).

relatively low product yields, ∼20-25% after preparative HPLC, when peaks were collected conservatively, as just the central peak fraction. Aggregates (∼12%) were observed in the conjugate prepared by the 2-IT method during long term storage (50 days) at 4 °C, whereas no aggregation was observed in conjugate prepared by the MEA method during storage. Interestingly, monomeric conjugate prepared using the 2-IT method eluted at an earlier HPLC retention time (9.5 min) than conjugate prepared using MEA (9.7 min) (Figure 2A), suggesting formation of an apparently larger molecular weight or differently shaped compound using the 2-IT method. To prove binding properties of StAv-MN-14s were retained, unlabeled conjugates were separately treated with WI2, bio-HSA-gal, and 111In-bio-DTPA followed by CEA. In each instance, the StAv-MN-14 showed complexation to these agents with the formed complexes eluting at appropriate retention times on SE-HPLC (Figure 2B). Retention of full biotin binding capacity was

imidate:protein ratio in galactosylation

MW by MALDI-MS

no. of galactose/WI2

0 15 30 75 400 400

149 562 149 830 150 517 153 012 157 740 160 000

0 1.1 4.0 14.6 34.7 44

shown by reference to unsubstituted streptavidin. Conjugates prepared by both the 2-IT and MEA reactions showed retention of four biotin binding sites (Table 1), whereas the StAv-biotin-MN-14 conjugate prepared using one of the biotin binding sites on streptavidin to link to biotinylated MN-14 showed 3 ( 0.5 biotin binding sites remaining, as would be expected (40). StAv-MN14 conjugate (25 µg) incubated in 250 µL of mouse serum also showed full retention of biotin binding capacity by SE-HPLC when aliquots of it at 0, 1, and 24 h were mixed with excess 111In-bio-peptide-DTPA. The biodistribution of StAv-MN-14 (prepared with MEA) was compared to that of MN-14 in GW-39 tumorbearing animals. Both were radioiodinated using the chloramine-T procedure and shown to bind to WI2 and CEA at 90-100% levels in vitro (data not shown). 125IStAv-MN-14 (10 µCi, 1 µg) and 131I-MN-14 (25 µCi, ∼2 µg) were coinjected into groups of tumor-bearing nude mice with major organs obtained 3, 24, and 72 h postinjection (Figure 3). Similar, but not identical, biodistributions for MN-14 and StAv-MN-14 were seen. Clearing Agents. The percentage of protein lysine residues that were modified with galactose was determined using a fluorescamine assay. Fluorometric analyses showed >81% modification of the available lysine residues on HSA with a 300-fold molar excess of thioimidate and 37-52% modification with a 100-fold molar excess of thioimidate. The WI2 samples were analyzed by MALDI-MS to determine the average level of modification per WI2. To determine the number of galactose residues attached to WI2, the molecular mass difference between the modified and unmodified WI2 was determined and divided by 236 (galactose residue formula weight). Results are shown in Table 2. HPLC analyses of mixtures of galactosex-WI2 (where x ) 0, 1, 4, 14.6, 35, and 44 residues) and MN-14 at a 1:1.6 molar ratio showed complete binding of galactosylated WI2 with MN14 (results not shown). Galactosylated WI2 also showed complete binding to the StAv-MN-14 conjugate, as did biotin-HSA-galactose, a third possible clearing agent (Figure 2B). In order to correlate the level of galactose substitution with blood clearance, WI2s with various galactose substitutions (0, 1, 4, 14.6, and 44) were radioiodinated and injected into BALB/c mice. Blood was drawn 1 h postinjection, and animals were sacrificed at 24 h for organ distribution studies. The results (Figure 4) showed rapid blood clearance of WI2 at galactose levels of >14. At these higher levels, the WI2 that was cleared from blood was taken up in the liver. At the highest galactose substitution, 44 per WI2, the blood level at 1 h was reduced to 0.7 ( 0.2% ID/g. Since high galactose


Karacay et al.

Figure 3. Comparison of the biodistribution of 125I-StAv-MN-14 (IgG) and 131I-MN-14 (IgG) in tumor-bearing mice in major organs: tumor, significant differences at all time points (p < 0.02); liver, significant differences at 4 and 72 h (p < 0.006); blood, significant differences at 24 and 72 h (p < 0.05); and kidney, significant differences at all time points (p < 0.02). Tumor sizes and standard deviations are shown in braces, and parentheses, respectively.

Figure 4. Effect of galactose levels on clearance of 125I]-galWI2. Higher galactose levels correspond to faster blood clearance.

substitutions did not effect WI2 binding to StAv-MN14, but provided faster clearance from the blood, the higher substitution level was used for the pretargeting studies in animals. The stability of the linkage between biotin and HSA toward mouse serum enzymes was examined in vitro and in vivo. 111In-Bz-DTPA-StAv-MN-14 showed a shift to a higher MW on a SE-HPLC column when biotin3HSA-gal39 incubated for 4 h in nude mouse serum was mixed with it, indicating the stability of biotin(s) on HSA to serum enzymes. One group of nude mice were injected with 200 µg of biotin3-HSA-gal39 and the other with 200 µg of biotin-HSA. Serum collected from the nude mice 1 and 8 h later was incubated with 111In-Bz-DTPAStAv-MN-14. HPLC analysis showed no binding with the biotin3-HSA-gal at 1 and 8 h, suggesting rapid blood clearance of the agent reduced its concentration in the blood to undetectable levels. Thus it was not possible to

test the in vivo stability of the biotin-HSA-gal. However, 111In-Bz-DTPA-StAv-MN-14 mixed with serum samples from mice given nongalactosylated biotin-HSA, at both 1 and 8 h, showed a shift to a higher molecular weight, thus demonstrating in vivo stability of at least one biotin-HSA linkage. These results suggest that the biotin-HSA-gal was also stable in the blood, particularly given its very short blood half-life. Biotinylated Chelating Agents. Biotinylated chelating agents were radiolabeled with 111In and mixed with unlabeled streptavidin prior to SE-HPLC to prove the presence of covalently attached biotin. Radiolabeled biotin- chelates eluted at 14-15 min, and streptavidin eluted at 11.5 min. Binding to streptavidin was demonstrated when the streptavidin and biotin-chelate mixture eluted at 11.5 min. Our first biotinylated chelate, biotin-DTPA, although stable in aqueous media, showed instability toward serum enzymes such that after 10 min in human serum only 58% of the radiolabeled chelate could still bind to streptavidin. At 21 h, no binding to streptavidin was observed. ITLC analysis of the serum sample at 21 h showed that the radiolabel was still bound to a chelate but had lost its biotin binding function. A similar behavior was observed with the first biotinDOTA derivative. In contrast, in vitro stability studies of biotinylated DOTA and Bz-DTPAs, wherein the biotin and chelate are linked through D-amino acids, consistently showed serum stability for 48 h (data not shown). In vivo stability was shown by examining the excreted product in the urine taken from tumor-bearing mice given 200 µg of StAv-MN-14 followed 48 h later by a clearing agent, 200 µg of biotin3-HSA-gal39, and then given 40 µCi (6 µg) of biotin-D-Phe-D-Lys-111In-Bz-DTPA-NH2 2 h later. Urine was collected 4 h after biotin-D-Phe-DLys-111In-Bz-DTPA-NH2 injection and analyzed by HPLC



in the blood than the DTPA complex at 1, 3, and 24 h. Most of the normal tissues had similar uptake with both complexes, except the liver and spleen. For example, at 24 h, the DOTA complex was about 3-fold higher (p ) 0.0028) in the liver (0.03 ( 0.006 vs 0.01 ( 0.002). Tumor uptake was higher than that in most organs, except the kidney. Even without pretargeting, tumor:blood ratios were as high as 10.6 ( 2.8 for the DTPA complex at 1 h but decreased to about 4:1 at 3 and 24 h. The tumor: blood ratio for the DOTA complex was about 5:1 at each time point over the first 24 h.

(A)

(B)

DISCUSSION

111In-bio-D-Phe-D-Lys(ITC-Bz-

Figure 5. In vivo stability of DTPA)-NH2. (A) HPLC of mouse urine collected 4 h after injection of 111In-labeled biotin in tumor-bearing mice pretargeted with StAv-MN-14 and cleared with bio3-HSA-gal 48 h later. 111In-labeled biotin was injected 2 h after the clearing agent. (B) The same mouse urine treated with streptavidin.

before and after mixing with streptavidin. A shift in the retention time from that of radiolabeled peptide (15 min) to that of the radiolabeled peptide-streptavidin complex (11.5 min) was observed (Figure 5). The biodistributions of two different biotinylated chelates were compared. 111In-labeled DOTA and Bz-DTPA derivatives of bio-D-Phe-D-Lys-NH2 (40 µCi; 5.8 × 10-9 moles of each) were injected intravenously in female nude mice bearing 5-day-old GW-39 tumors (average weight of 0.04-0.05 g). Figure 6 shows the biodistribution of these agents over a 48 h time period with five animals per group per time interval. Each conjugate was cleared from the blood very rapidly with less than 0.1% per gram at 1 h. The DOTA complex was 2-fold higher (p < 0.05)

This article describes reagent validation work which we considered essential before formal two-step pretargeting studies using a biotin/streptavidin recognition system, due to the complexities involved in the system. Certain potential pitfalls were expected prior to embarking on the work, and the importance of others became apparent as we proceeded. Some of these primarily involve biological aspects such as doses and timings and will be discussed elsewhere (38). Chemistry considerations included the simple ability to separate a pure sample of StAv-MN-14 for testing, a demonstration (qualitative and quantitative) that it retained its binding capacities and that it targeted tumor, choice of clearing and biotin chelating agents, and suitable stability studies, prior to embarking on more expensive preclinical in vivo studies. For the first step, streptavidin conjugates of the MN14 mab were prepared after streptavidin was derivatized with sulfo-SMCC to introduce maleimide groups. In one method, lysine residues of MN-14 were modified with 2-IT to introduce free SH groups, and in the other, disulfide bonds on IgG were reduced with MEA. In both

Figure 6. Comparison of the bioistribution of 111In-labeled biotinylated chelates bio-D-Phe-D- Lys(DOTA)-NH2 and bio-D-Phe-DLys(Bz-DTPA)-NH2 in GW-39 tumor-bearing nude mice.


cases, varying reaction conditions were examined to improve conjugate yield, but conditions such as pH (5.17.4), concentration (4.6-11 mg/mL), temperature (4-37 °C), and reaction ratios (IgG:SA molar ratios of 0.5-4:1) did not improve yields significantly with either method. This is almost certainly due to the rapid kinetics of the thiol reaction with the maleimide under the range of conditions studied. Somewhat higher conjugate yields could be obtained, but these reactions invariably were accompanied by higher aggregate formation. To limit aggregate formation and protein losses, we chose to use a low number of sulfhydryl groups (2 ( 0.3 SH/IgG) generated on MN-14 using MEA, and the unused IgGSH/IgG was collected during purification and recycled. Aggregate formation was minimized, but never eliminated. Others (16, 28) have claimed high yields of streptavidin-mab conjugates, but only low yields were observed with both methods described here. No attempt was made to improve yields for StAv-biotin-MN-14, prepared according to the method of Hnatowich et al. (40). The difference in retention times (apparent MWs) of StAv-MN-14 conjugates prepared by the two methods could be due to the linkage chemistry (Figure 1). We speculate that conjugates prepared via internal mab disulfide bonds must be approached more closely by the reacting SA than those prepared using 2-IT. In either case, non-radiolabeled StAv-MN-14 conjugates retained binding properties to CEA, to all possible clearing agents (WI2, galactosylated WI2, and biotinylated HSA), and to the biotin-chelate conjugates prepared as final step agents. An important aspect of using StAv-MN-14 to pretarget tumors is the potential to increase uptake of radioactivity localized at tumor 4-fold due to the four biotin binding sites on streptavidin. For this to occur, it is essential that the biotin binding capacity on streptavidin be preserved after covalent attachment to MN-14. Biotin binding was found to be quantitative for both conjugates prepared with SMCC. In addition to stability in aqueous media, StAv-MN-14 conjugates were shown to retain biotin binding capacity in mouse serum in vitro for 24 h. StAv-MN-14 conjugate prepared using MEA was selected since it was found to be more resistant to aggregate formation during storage than the conjugate prepared using 2-IT. Radioiodinated StAv-MN-14 prepared using the chloramine-T procedure did retain binding to CEA and WI2 (90-100%) in vitro, but it showed a partial loss (2090%) of biotin binding capability to biotinylated HSA and other biotin compounds (results not shown), probably due to oxidation of biotin-binding tryptophan residues of the StAv, and made this type of agent unsuitable for dual radiolabel pretargeting work. Other iodination methods such as the Bolton-Hunter method were briefly investigated but discarded primarily due to unsatisfactory radioiodination yields. Instead, for radiotracer purposes, Bz-DTPA was attached to lysine residues of StAv-MN14, radiolabeled with 88Y without loss of binding characteristics, and used for targeting agent biodistributions. Clearance agents based on biotin and anti-idiotypic recognition of StAv-MN-14 were developed and were further modified with galactose for rapid blood clearance (29). Studies with galactosylated WI2s showed faster blood clearance at higher galactose substitutions (Figure 4). In the case of HSA, stable biotin(s) also needed to be attached as a recognition system, and our in vitro and in vivo stability studies showed the stability of at least one biotin-HSA linkage toward serum enzymes, such as biotinidases. We cannot rule out the fact that some biotin(s) might have been cleaved from the HSA, since

Karacay et al.

Figure 7. Metal complexes of biotinylated peptide chelating agents.

no effort was made to quantitate the biotins remaining bound on injected HSA. The galactosylation reaction was somewhat inconsistent, because different levels of galactose modification were observed with the same ratios of activated thioimidate:protein (Table 2). Thus, proteins were analyzed for galactose content after each such modification. Most interestingly, the level of galactose substitutions on either WI2 or HSA had no apparent effects on their respective ligand binding properties. A high galactosylation level was critical for fast blood clearance of either clearing agent (g35 galactose residues per WI2). We have decided to concentrate on WI2, thinking that anti-idiotypic clearance will be a superior mechanism due to its lack of competitive binding to the tumor-targeted first-step agent, lack of potential for blocking to StAv binding sites, and more complete serum clearance. For the final step, biotin-chelate conjugates consisting of biotin, a linker, and a chelating agent were prepared, first using commercially available [5-(biotinamido)pentyl]amine. Biotin-chelate conjugates with both DOTA and Bz-DTPA directly attached to this agent were found to be unstable toward serum enzymes. D-Amino acid peptides were selected as biotin-to-chelate linkers on the basis that they might offer stability toward serum enzymes and also later allow synthesis of reagents with variable hydrophobicities and serum clearance rates (hence, more tumor passes and higher tumor uptake). DOTA and Bz-DTPA were attached to a D-lysine side chain of the peptides and biotin to the R-amino terminus. All corresponding 111In-labeled species then showed good in vitro and in vivo stability. When 111In-labeled biotinpeptide-Bz-DTPA and bio-peptide-DOTA were compared for clearance in tumor-bearing mice (Figure 6), the former, containing a negatively charged 111In-DTPA complex (Figure 7), cleared blood faster. The first step in developing a complex delivery system such as this is to ensure that reagents are suitably characterized, functional, and stable under expected in vivo conditions, and this has been done. Investigation of a two-step pretargeting approach using the agents selected through these studies will be described elsewhere (38). Within this work, other issues then have to be considered, including the timing and dose of each reagent, the specific activity of the radiolabeled biotin-


chelate, and the effect of competition from endogeneous biotin for targeted StAv-MN-14 biotin-binding sites. ACKNOWLEDGMENT

We acknowledge Agatha Sheerin for assisting in reagent preparation and Rosarito Aninipot for animal biodistribution studies. This work was supported in part by U.S. Public Health Service Grants CA-37895 and CA39841 from the NIH. LITERATURE CITED (1) Bamias, A., and Epenetos, A. A. (1992) Two-step strategies for the diagnosis and treatment of cancer with bioconjugates. Antibody, Immunoconjugates, Radiopharm. 5, 385-395. (2) Goodwin, D. A., Meares, C. F., McCall, M. J., McTigue, M., and Chaovapong, W. (1988) Pre-targeted immunoscintigraphy of murine tumors with indium-111-labeled bifunctional haptens. J. Nucl. Med. 29, 226-234. (3) Stickney, D. R., Anderson, L. D., Slater, J. B., Ahlem, C. N., Kirk, G. A., Schweighardt, S. A., and Frincke, J. M. (1991) Bifunctional antibody: A binary radiopharmaceutical delivery system for imaging colorectal carcinoma. Cancer Res. 51, 6650-6655. (4) Hawkins, G. A., McCabe, R. P., Kim, C.-H., Subramanian, R., Bredehorst, R., McCullers, G. A., Vogel, C.-W., Hanna, M. G., Jr., and Pomata, N. (1993) Delivery of radionuclides to pretargeted monoclonal antibodies using dihydrofolate reductase and methotrexate in an affinity system. Cancer Res. 53, 2368-2373. (5) Bos, E. S., Kuijpers, W. H. A., Meesters-Winters, M., Pham, D. T., deHaan, A. S., van Doormalen, A. M., Kasperson, F. M., van Boeckel, C. A. A., and Gougeon-Bertrand, F. (1994) In vitro evaluation of DNA-DNA hybridization as a two-step approach in radioimmunotherapy of cancer. Cancer Res. 54, 3479-3486. (6) Hnatowich, D. J., Virzi, F., and Rusckowski, M. (1987) Investigations of avidin and biotin for imaging applications. J. Nucl. Med. 28, 1294-1302. (7) Paganelli, G., Pervez, S., Siccardi, A. G., Rowlinson, G., Deleide, G., Chiolerio, F., Malcovati, M., Scassellati, G. A., and Epenetos, A. A. (1990) Intraperitoneal radiolocalization of tumors pre-targeted by biotinylated monoclonal antibodies. Int. J. Cancer 45, 1184-1189. (8) Axworthy, D. B., Theodore, L. J., Gustavson, L. M., and Reno, J. M. (1993) Pretargeting methods and compounds. International Patent Application WO 93/25240. (9) Axworthy, D. B., Fritzberg, A. R., Hylarides, M. D., Mallet, R., Theodore, L. J., Gustavson, L., Su, F.-M., Beamuier, P. L., and Reno, J. M. (1995) Durable complete remissions of breast, lung and colon tumor xenografts with a single dose of pretargeted Y-90 in a mouse model. J. Nucl. Med. 36, 217P (abstract). (10) Saga, T., Weinstein, J. N., Jeong, J. M., Heya, T., Lee, J. T., Le, N., Paik, C. H., Sung, C., and Neuman, R. D. (1994) Two-step targeting of experimental lung metastases with biotinylated antibody and radiolabeled streptavidin. Cancer Res. 54, 2160-2165. (11) Sung, C., van Osdol, W. W., Saga, T., Neumann, R. D., Dedrick, R. L., and Weinstein, J. L. (1994) Streptavidin distribution in metastatic tumors pretargeted with a biotinylated monoclonal antibody: Theoratical and experimental pharmacokinetics. Cancer Res. 54, 2166- 2175. (12) Marshall, D., Pedley, R. B., Melton, R. G., Boden, R., and Begent, R. H. J. (1995) Galactosylated streptavidin for improved clearance of biotinylated intact and F(ab’)2 fragments of an anti-tumor monoclonal antibody. Br. J. Cancer 71, 18-24. (13) Paganelli, G., Chinol, M., Grana, C., De Cicco, C., Cremonesi, M., Meares, C. F., Franceschini, R., Tarditi, L., and Siccardi, A. G. (1995) Optimization of the three-step pretargeting approach for diagnosis and therapy in cancer patients. J. Nucl. Med. 36, 225P (abstract). (14) Breitz, H., Ratliff, B., Reno, J., Axworthy, D., Fritzberg, A., Su, F.-M., Appelbaum, J., Kunz, S., Seiler, C., and Weiden,

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