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Basic Science Investigation |
1 Research Laboratories, Schering AG, Berlin, Germany; 2 Philogen S.p.A, Siena, Italy; 3 Department of Experimental and Clinical Immunology, Istituto Giannina Gaslini, Advanced Biotechnology Center, Genoa, Italy; and 4 Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, Zürich, Switzerland
Correspondence: For correspondence or reprints contact: Dietmar Berndorff, PhD, Schering AG, Müllerstrasse 178, D-13342 Berlin, Germany. E-mail: dietmar.berndorff{at}schering.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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-Lys-NH2 residues of the molecule to allow for a direct chelator-based labeling with 99mTc. Tumor-targeting, pharmacokinetic, and scintigraphic imaging properties of the radiolabeled scFvs were evaluated in nude mice bearing murine F9 teratocarcinoma. Results: 99mTc labeling of the L19 derivatives yielded radiochemically pure proteins maintaining high immunoreactivity to ED-B fibronectin, as measured by affinity chromatography. Size-exclusion high-performance liquid chromatographic analysis of labeled L19 derivatives demonstrated either dimeric species (L19-His) or a mixture of predominantly associative dimeric and monomeric species (AP39, L19-Hi20). 99mTc-AP39 showed the most favorable biodistribution and imaging properties with high and fast tumor uptake (8.3 percentage injected dose per gram at 3 h after injection), rapid blood clearance and renal excretion, leading to high signal-to-noise ratios (tumor-to-blood ratio of 6.4 at 3 h after injection), and excellent planar scintigraphy in vivo. Conclusion: ED-B fibronectin can be efficiently targeted by 99mTc-AP39 and scintigraphically visualized in tumor-bearing mice, providing a potentially useful clinical tool for imaging of angiogenesis-associated ED-B fibronectin–expressing human tumors.
Key Words: molecular imaging • scintigraphy • ED-B fibronectin • angiogenesis • 99mTc labeling
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Fibronectin is a universal cell-adhesion molecule and represents the most widely distributed extracellular matrix protein. The splice variant ED-B fibronectin, containing the extra domain B, is an oncofetal antigen expressed at different levels in the stroma associated with the neovasculature of solid tumors and, thereby, known as a marker of angiogenesis (3). Its expression is restricted to angiogenic processes in embryonic development and tumorigenesis. In contrast, it is undetectable in most mature vessels and normal adult tissues, except for tissues undergoing physiologic remodeling, such as endometrium and ovaries (4,5). High levels of ED-B expression can be detected in primary tumors as well as metastatic lesions of almost all human solid cancer entities, including breast, colorectal, and non–small cell lung cancer (6–8). Therefore, targeting ED-B fibronectin for in vivo imaging purposes has the potential to serve not only as a general early tumor marker in tumorigenesis but also play a role as a surrogate parameter for monitoring and predicting the success of antiangiogenic cancer therapy.
L19 is a single-chain antibody fragment (scFv) that specifically targets the ED-B sequence of fibronectin and was first identified by Pini et al. (9). L19 was isolated from a phage display library of a synthetic human antibody repertoire binding to ED-B fibronectin with high affinity in the subnanomolar range (dissociation constant = 54 pmol/L). Specific accumulation of 125I-L19 around tumor neovasculature and in tumor stroma correlated with ED-B fibronectin expression as subsequently demonstrated by microautoradiography analysis (10). 125I-L19 showed specific tumor targeting combined with a fast blood clearance (10,11) in mice. Recently, 123I-L19 was used for scintigraphic detection of ED-B fibronectin in cancer patients (8). In this study it was shown that 123I-L19 selectively localized in tumor lesions of aggressive lung cancer as well as in liver metastases of colorectal cancer patients, directly correlating with the ED-B fibronectin expression. However, the use of radioactive iodine for in vivo imaging—except for thyroid imaging with iodide—is mainly hampered by the inconvenient labeling procedure (i.e., on-site preparation using kit chemistry is not feasible), the high costs, and the chemical instability due to dehalogenation. Therefore, we wished to introduce 99mTc as a more advantageous isotope for scintigraphic imaging into the protein. 99mTc is characterized by optimal physical properties, such as a half-life of 6 h, a nearly monoenergetic 
-emission of 140 keV, and its instant availability through a generator system.
Herein we describe the synthesis, 99mTc labeling, and in vivo characterization of the scFv L19 with altered labeling positions and radiometal chelating moieties. Because of the thiophilic nature of Tc(V), a free sulfhydryl group had to be introduced into the protein sequence for stable radiometal binding. Therefore, the amino acid sequence (Gly)3-Cys-Ala was genetically inserted at the C terminus of L19, resulting in a recombinant protein named AP39, which is suitable for the direct labeling with 99mTc. In this molecule, technetium can form a Tc(V) oxo metal complex with, for example, 3 amide nitrogen atoms and 1 thiol sulfur atom in the Gly-Gly-Cys sequence (12) as 1 possible coordination mode. In a second approach, a (His)6 tag was genetically introduced (L19-His) at the C terminus of the protein for direct 99mTc labeling with a Tc(I) carbonyl core (13). The Tc(V)O-core is also known to form stable complexes with mercaptoacetylglycine derivatives (MAG2 or MAG3), which can be conjugated efficiently to biomolecules (14). Hence, in a third approach, L19 was chemically modified by a bifunctional MAG2-type chelator (Fig. 1), conjugated to the -amino groups of Lys-residues of the scFv (L19-Hi20), and radiolabeled with 99mTc.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Radiolabeling with 99mTc
General Procedures.
All chemicals were purchased from Fluka, Aldrich, or Merck and were used without further purification. Sodium pertechnetate 99mTc was obtained on the day of use from an ElumaticIII-8 99Mo/99mTc-generator (CIS-Bio-International). High-performance liquid chromatographic (HPLC) analysis of radiolabeled L19 derivatives was performed on a Biotek system (BioTek Instruments), equipped with a radioflow HPLC detector (EG & G Bertold). Samples were injected onto TSK-Gel size-exclusion columns (Tosoh Haas) and eluted with buffer (0.1 mol/L Na2HPO4, 0.1 mol/L Na2SO4, 0.05% NaN3, pH 6.7) at a flow rate of 0.4 mL/min. Elution was monitored by ultraviolet detection at 275 nm (100 µL) and -detection (10 µL). A radioisotope dose calibrator (PTW-Dr. Pychlau GmbH) was used to determine the radioactive content of the labeling solutions. Small radioactive samples were counted on a -counter (Compugamma; LKB Wallac).
99mTc Radiolabeling of AP39.
Before radiolabeling, AP39 had to be reduced by adding 83.3 µL Tris-(2-carboxyethyl)phosphine-hydrochloride (TCEP) solution (14.34 mg TCEP·HCl in 5 mL of 0.1 mol/L Na2HPO4 buffer, pH 7.4) to a solution of 400 µg purified AP39 in 140 µL phosphate-buffered saline (PBS)/10% glycerol (pH 7.4). The reaction mixture was gently shaken for 1 h at room temperature. TCEP was removed from the AP39 preparation by chromatography using a NAP-5 column (Sephadex G-25 DNA grade, eluent: PBS; Amersham Biosciences). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the isolated product confirmed the quantitative transformation of S–S-dimeric AP39 to AP39 in the SH-monomer and associative dimer form. Protein yields of 136 µg/180 µL PBS could be achieved. For radiolabeling, 4.2 mg disodium-L-tartrate were placed in a 2-mL glass-vial followed by addition of 136 µg reduced AP39 in 180 µL PBS. The solution was diluted with 100 µL aqueous Na2HPO4 buffer (1 mol/L, pH 10.5) and 100 µL 99mTc generator eluate plus 10 µL tin-II chloride solution (5 mg SnCl2, 1 mL of 0.1 mol/L HCl) were added. The reaction mixture was shaken for 30 min at 37°C. 99mTc-AP39 was purified by gel chromatography using a NAP-5 column (Sephadex G-25 DNA grade, eluent: PBS). The 99mTc-compound was protected against air oxidation by adding 2 µL tin-II chloride solution (5 mg SnCl2, 1 mL of 0.1 mol/L HCl). The radiochemical yield and purity were determined by SE-HPLC. Therefore, a Tosoh Haas, TSK-Gel SWxL2000 SE column (300 x 7.8 mm) was eluted with an isocratic flow (0.5 mL/min) of aqueous Na2HPO4/Na2SO4 (0.1 mol/L) buffer solution (1:1, v/v), completed with 0.05% NaN3 (pH 6.7).
99mTc Radiolabeling of L19-His.
Radiolabeling of L19-His was performed by applying the tricarbonyl method as described earlier (14) using [99mTc(OH2)3(CO)3]+. Quality control of the synthesized [99mTc(OH2)3(CO)3]+ was performed by gradient HPLC, injecting 20 µL (2 MBq/mL) on a Eurospher 100-C18 column (250 x 4 mm; Knauer GmbH) and eluting with a gradient of 0.05 mol/L triethylammoniumphosphate (pH 2.25) (A) and MeOH (B) (0% B to 100% B within 20 min). To radiolabel L19-His with [99mTc(OH2)3(CO)3]+, 100 µg of the protein (1.0 mg/mL scFv L19-His in 10 mmol/L N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) buffer, pH 7.5, 150 mmol/L NaCl) were added to 200 µL PBS and 37 MBq [99mTc(OH2)3(CO)3]+ solution (31 µL). The mixture was incubated for 1 h at 37°C and the 99mTc-L19-His product was purified by affinity chromatography using a NAP-5 column (eluent: PBS). The radiolabeled compound was analyzed by SE-HPLC as described.
99mTc Radiolabeling of L19-Hi20.
The chelator Hi20 (2-tert-butyldisulfanyl-N-({[(4-isothiocyanatobutylcarbamoyl)methyl]carbamoyl}-methyl)acetamide) was conjugated to -NH2 groups of L19 by dissolving 2 mg of L19 in 1.3 mL PBS and dialyzing twice with sodium borate buffer (200 mL, 0.1 mol/L, pH 8.5) in a Slide-A-Lyzer (equipped with a 10-kDa molecular weight cutoff membrane; Pierce Chemicals). Hi20 (1.5 mg) was dissolved in N,N-dimethylformamide (150 µL) and added to the protein solution. The mixture was incubated for 3 h at 37°C. The solution was allowed to cool to room temperature for 10 min and was then dialyzed with PBS (400 mL, 0.1 mol/L, pH 7.4) 3 times for 1 h and once overnight. Before radiolabeling, the protein–chelator conjugate was reduced by the addition of TCEP (150 µL, 0.1 mol/L, pH 7.4) and incubation at 37°C for 1 h The sample was purified on a NAP-5 column, preeluted with PBS/0.5% bovine serum albumin (BSA), and, finally, eluted with PBS (3 x 3 mL). For radiolabeling, 110 µg L19-Hi20 (in 310 µL PBS) were supplemented with 3.3 mg sodium tartrate dihydrate, phosphate buffer (100 µL, 1.0 mol/L, pH 10.5), 99mTc generator eluate (70 MBq, 50 µL, sodium pertechnetate (VII)), and stannous (II) chloride (10 µL, stock solution: 5 mg SnCl2 in 1 mL of 0.1 mol/L HCl). The mixture was incubated at 37°C for 30 min and purified on a NAP-5 column that was preeluted with PBS/0.5% BSA. The product was finally eluted with PBS (3 x 3 mL). An addition of 5 µL of stannous (II) chloride solution completed the procedure to stabilize the product. Analysis followed by SE-HPLC.
Immunoreactivity
Assessment of immunoreactivity of 99mTc-L19 derivatives was performed by affinity chromatography according to Tarli et al. (10) using a column containing ED-B fibronectin–conjugated Sepharose.
Cell Lines and Animals
Mouse embryonal teratocarcinoma cells (F9) were purchased from the American Tissue Culture Collection. The tumor cells were cultivated in Dulbecco's modified Eagle medium with Glutamax (Invitrogen), supplemented with 10% fetal calf serum (Invitrogen) and maintained at 37°C in 5% CO2 in air. To induce tumors, female nude mice (NMRI nu/nu; Taconic), weighing 20–25 g, were injected subcutaneously with 1 x 106 F9 cells in a volume of 100 µL PBS with Ca2+/Mg2+ (Invitrogen) into the right hind limb. After 11 d, tumors reached a size of approximately 0.8 cm in diameter and the animals were used for biodistribution and imaging experiments. All animal experiments were performed in compliance with the current version of the German law on the Protection of Animals.
Biodistribution Studies
Tumor-bearing mice were injected intravenously in the tail vein with approximately 74 kBq of 99mTc-L19 derivatives. Three mice per time point were sacrificed at 15 min and at 1, 3, 5, and 24 h after injection for organ excision. In addition, urine and feces were collected over time. The dissected organs and collected excretions were counted for radioactivity in a -counter (Compugamma; LKB Wallac), and values of the percentage injected dose per gram (%ID/g) and the percentage injected dose (%ID) were calculated.
Imaging
Tumors were imaged when they reached an area of 80–100 mm2. For imaging, conscious mice were injected intravenously in the tail vein with 4–7 MBq of 99mTc-L19 derivatives. The mice were anesthesized using subcutaneously injected rompun/ketamin (2:1 mL/kg) and images of each animal were taken at 5 and 24 h after injection. Imaging was performed using an SP-4 HR -camera (Elscint) with the following imaging parameters: energy, 140 keV; rate mode, normal; collimator, 4; static; frame size, 256; zoom, 2; rotation, 180°; view, posterior.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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99mTc Labeling of L19 Derivatives
99mTc Labeling of AP39.
Immediately before radiolabeling, the purified AP39 was reduced with TCEP. The radiolabeling procedure, using the purified and TCEP-reduced AP39, with 99mTc gave radiochemical yields of >50% (NAP-5 column purified). A radiochemical purity of >95% was observed, as determined by SE-HPLC analysis. The product is present as a mixture of predominantly dimeric (
81%) and monomeric (16%) 99mTc-AP39, as determined by SE-HPLC (Fig. 3). ED-B affinity chromatography revealed an immunoreactivity of 96%. The specific activity of 99mTc-AP39 was 35 MBq/nmol (dimer).
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99mTc Labeling of L19-Hi20.
99mTc labeling of L19 via the bifunctional chelator Hi20 resulted in a radiochemical yield of 54% and a radiochemical purity of >91%. The specific activity was 21 MBq/ nmol (dimer). NAP-5 purification yielded 99mTc-19-Hi20 as a monomer/dimer mixture, as monitored by SE-HPLC (Fig. 3). However, a complete separation of the dimeric and monomeric format could not be achieved by this HPLC method. The immunoreactivity was 82%, as measured by ED-B affinity chromatography.
Biodistribution Data
Biodistribution experiments with the 99mTc-L19 derivatives AP39, L19-His, and L19-Hi20 were performed over 24 h in nude mice bearing the murine teratocarcinoma F9 (Tables 1–3
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Since the introduction of the anti-ED-B fibronectin scFv L19, several therapeutically active L19 conjugates have been investigated. For example, the fusion protein L19-TNF-
(TNF- is tumor necrosis factor ) showed substantial inhibition of tumor growth in mice (16), and the cytokines interleukin 12 and TNF- fused to the L19 protein demonstrated in combination the complete cure of tumor-bearing mice with only minor side effects (17). The potential therapeutic use of L19 derivatives may trigger a clinical need of a diagnostic counterpart for imaging.
The targeting of ED-B fibronectin for imaging has already been investigated in a preliminary clinical study using 123I-L19. Recurrent glioblastoma, small cell lung cancer, and a large liver metastasis of a colon carcinoma could be detected using the iodinated scFv (8). However, for clinical use, 99mTc is the preferred isotope compared with 123I because of its shorter half-life (6 h), the convenient labeling procedure, and the optimal -energy. Being generator produced, 99mTc is inexpensive and readily available. If considering L19 as a targeting molecule, 99mTc offers the additional advantage that its physical half-life matches ideally the biologic half-life of the scFv. The creation of a 99mTc binding site by the conjugation of a chelator to the targeting molecule can be associated with instability of the labeled proteins (18), whereas attempts to genetically engineer cysteine groups into proteins may lead to problems with protein expression, folding, or stability (19). We have evaluated the direct and indirect 99mTc labeling approaches of L19, followed by in vivo investigations of tumor-targeting potential and pharmacokinetic behavior. Direct radiolabeling of L19 with technetium has been performed after creation of an intramolecular 99mTc binding site via genetically inserting the amino acid sequence (Gly)3-Cys-Ala (AP39) or the (His)6 domain (L19-His). Both derivatives could be expressed in E. coli in amounts that were sufficient for the studies described herein (i.e., 0.5 mg/L for AP39 and 5 mg/L for L19-His). For both AP39 and L19-His recombinant scFvs, the site-directed binding of 99mTc did not lead to a loss in immunoreactivity (>89%). As an alternative to the intramolecular direct labeling with 99mTc (AP39, L19-His), we have evaluated the introduction of a bifunctional chelator that was attached at -NH2 groups within the molecule. Such conjugated bifunctional chelators may bind to or interfere with regions of the antibody that are mandatory for antigen recognition. Thereby, immunoreactivity and tumor-targeting potential of the radioimmunoconjugate could be adversely affected (20). However, the 99mTc labeling of L19-Hi20 resulted in radiometal chelate conjugates of high immunoreactivity (>80%).
Because the affinity as well as the resulting in vivo targeting properties of antibody fragments may be improved for covalent and associative dimers compared with monomeric derivatives (21,22), the formats of our labeling products were evaluated. The scFv L19 has already been described as a mixture of monomer and homodimer (23). The 99mTc-L19 derivatives investigated in this study appeared either as a monomer/dimer mixture (99mTc-AP39, 99mTc-L19-Hi20) or as a completely dimeric product (99mTc-L19-His).
F9 tumors (murine teratocarcinoma) were described to express high levels of ED-B fibronectin (11,23) and, therefore, have been used as a tumor model to investigate targeting by L19 and derivatives thereof (16,24,25). Biodistribution experiments showed specific tumor uptake of all 99mTc-L19 derivatives in F9-bearing mice combined with a rapid blood clearance. Elevated uptake in the ovaries and the uterus was regarded as specific targeting of ED-B fibronectin, which is physiologically expressed in reproductive organs (4,5). 99mTc-AP39 demonstrated high tumor uptake, fast excretion, and low background activity comparable with the in vivo data of 125I-L19 in the same tumor model (10,11). The resulting tumor-to-tissue ratios (e.g., T/B ratio of 6.3 at 3 h after injection) were even slightly better than those of 125I-L19 (e.g., T/B ratio at 3 h after injection of 1.9 (10) and 1.7 (11)). The same observation was made by Kang et al. (26), who found higher tumor-to-tissue ratios of 99mTc-scFvs against a high-molecular-weight melanoma–associated antigen compared with 125I-labeled derivatives of these scFvs, with the exception of the kidneys. In comparison, other 99mTc-scFvs—for example, against carcinoembryonic antigen—were reported to have much lower T/B ratios of only 4 after 24 h (27) or of 2.07 at 4 h after injection into tumor-bearing mice (28). Scintigraphic images of F9 tumors by 99mTc-AP39 can be achieved more rapidly and with a better quality compared with fluorescent images obtained by Cy7-labeled L19 (25).
99mTc-L19-His showed a tumor-targeting potential comparable with that of 99mTc-AP39 but showed an elevated accumulation of radioactivity in nontarget organs. Its high kidney accumulation is an especially critical issue, resulting in unfavorable background activity in images after 3 and 5 h. High accumulation of radioactivity in the kidneys with His-tagged scFvs when radiolabeled with [99mTc(OH2)3(CO)3]+ was already described by Waibel et al. (29), who found, for an anti–c-erb B2 scFv, a kidney uptake of 108.6 %ID/g after 24 h.
The in vivo targeting properties of 99mTc-L19-Hi20 were comparable with those of 99mTc-AP39, as demonstrated by a similar tumor uptake. However, its slower blood kinetics resulted in a high background activity, leading to inferior image quality. This effect was presumably caused by a larger amount of high-molecular-weight impurities as well as the chelator Hi20. It has been suggested that the pharmacokinetic behavior with regard to half-life in the blood and accumulation in nontarget organs may be strongly influenced by chelators conjugated to the protein (20).
Besides tumor retention, the most prominent uptake for all three investigated L19 derivatives was observed in the kidney. High kidney uptake is a common observation together with antibody fragments radiolabeled with 99mTc, 111In, or 90Y. Whereas 125I-labeled proteins are known to be rapidly deiodinated in the kidney (30), 99mTc-scFvs seem to be temporarily trapped by the renal tubular cells after endocytosis (31). Besides the isotope itself, kidney uptake of antibody fragments is influenced by the chelator and the linker conjugated to the protein (32). However, in contrast to therapy, high uptake in the kidneys is not as problematic because today SPECT enables separation of closely grouped body areas.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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References |
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