P. August Schubiger

Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex.

We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99mTc(I)–carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.

Metal carbonyl syntheses XXII. Low pressure carbonylation of [MOCl4]− and [MO4]−: the technetium(I) and rhenium(I) complexes [NEt4]2[MCl3(CO)3]☆

Low pressure carbonylation (1 atm) of [MOCl4]− or [MO4]− in the presence of BH3-tetrahydrofuran and halides X− results in the clean formation of [NEt4]2[MX3(CO)3] containing monovalent rhenium or technetium (MRe or Tc; X Cl or Br). In the case of the radioactive element technetium this low pressure synthesis is an important progress since potential hazards of traditional high pressure carbonylations are thus avoided. The complex [NEt4]2[ReBr3(CO)3] crystallizes in the space group P1 with a = 10.166(2), b = 16.393(4), c = 17.243(5) A, and α = 69.27(20, β = 86.42(2), γ = 88.61(2)°, Z = 4 and V = 2682(1) A3. [NEt4]2[MX3 (CO)3] is a versatile precursor compound of other Re(I) and Tc(I) complexes; substitution of the halide ligands by a variety of σ-donor ligands is facile even under mild conditions. Examples include reactions with CNtBu to yield quantitatively TcCl(CNtBu)2(CO)3 and with a tetradentate phosphine ligand to yield the dinuclear complex [Re2Br2(CO)6(tetraphos)]. The x-ray structures of both compounds were determined. Dissolution of [NEt4]2[MX3(CO)3] in water under aerobic conditions results in the unusual organometallic aqua cation [(M(OH2)3(CO)3] +; this species is stable in water for days (IR spectroscopy).

Synthesis and reactivity of [NEt4]2[ReBr3(CO)3]. Formation and structural characterization of the clusters [NEt4][Re3(µ3-OH)(µ-OH)3(CO)9] and [NEt4][Re2(µ-OH)3(CO)6] by alkaline titration

The dianionic rhenium(I) complex [ReBr3(CO)3]2– has been synthesized and characterized. This complex is an important starting material for compounds containing the fac-Re(CO)3 moiety since the three bromide ligands are very weakly bound. Particularly in co-ordinating solvents, the bromides are substituted by solvent molecules thus generating the strong 12e– Lewis acid [Re(H2O)3(CO)3]+2. This formula was confirmed, particularly in water, by IR spectroscopic methods. Complex 2 is stable in aqueous solution even when exposed to air for weeks. It has been titrated by NaOH solutions. The change in pH was detected potentiometrically and found to occur very slowly, indicating a reaction other than protonation deprotonation. Depending on the rate of titration and the total concentration of OH–, the two rhenium(I) hydroxo complexes [NEt4][Re3(µ3-OH)(µ-OH)3(CO)9] and [NEt4][Re2(µ-OH)3(CO)6] were isolated in good yield and structurally characterized. The former crystallizes in the orthorhombic space group Pnma with a= 10.752(7), b= 13.783(8) and c= 18.254(12)A. The skeletal framework is that of a cube lacking the Re(CO)3 unit in one corner. The latter crystallizes in the orthorhombic space group P2mm with Z= 1, a= 6.551(2), b= 7.413(2) and c= 12.084(2)A. The two Re(CO)3 moieties are bridged by three OH– ligands which create a mirror plane. A second mirror plane is perpendicular to it along the Re-Re axis.

Efficient Inhibition of Intra-Peritoneal Tumor Growth and Dissemination of Human Ovarian Carcinoma Cells in Nude Mice by Anti-L1-Cell Adhesion Molecule Monoclonal Antibody Treatment

The L1 cell adhesion molecule is implicated in the control of proliferation, migration, and invasion of several tumor cell types in vitro. Recently, L1 overexpression was found to correlate with tumor progression of ovarian carcinoma, one of the most common causes of cancer-related deaths in gynecologic malignant diseases. To evaluate L1 as a potential target for ovarian cancer therapy, we investigated the effects of anti-L1 monoclonal antibodies (chCE7 and L1-11A) on proliferation and migration of L1-positive human SKOV3ip ovarian carcinoma cells in vitro and the therapeutic efficacy of L1-11A against i.p. SKOV3ip tumor growth in nude mice. In vitro, both anti-L1 antibodies efficiently inhibited the proliferation of SKOV3ip cells as well as other L1-expressing tumor cell lines (renal carcinoma, neuroblastoma, and colon carcinoma). On two cell lines, hyper-cross-linking of L1-11A with a secondary antibody was necessary for significant inhibition of proliferation, indicating that cross-linking of L1 is required for the antiproliferative effect. L1-negative prostate carcinoma cells were not influenced by antibody treatment. Biweekly treatment of ovarian carcinoma-bearing mice with L1-11A led to a dose-dependent and significant reduction of tumor burden (up to -63.5%) and ascites formation (up to -75%). This effect was associated with reduced proliferation within the tumors. L1-directed antibody-based inhibition of peritoneal growth and dissemination of human ovarian carcinoma cells represents important proof-of-principle for the development of a new therapy against one of the leading gynecologic malignant diseases.

Reactions with the technetium and rhenium carbonyl complexes (NEt4)2[MX3(CO)3]. Synthesis and structure of [Tc(CN-But)3(CO)3](NO3) and (NEt4)[Tc2(μ-SCH2CH2OH)3(CO)6]

Abstract Starting from [MO4]− (M = Tc, Re) the low-pressure (1 atm.) synthesis of the important starting material (NEt4)2[MCl3(CO)3] could be improved in terms of yield and time. These MI complexes were used for the preparation of compounds containing the “fac-M(CO)3” moiety under ambient conditions. The substitution with the isocyanide ligand CN-But is described. In the case of rhenium, IR spectroscopical investigations in the CO stretching region allowed the observation of the stepwise formation of [Re(CN-But)3(CO)3]+. It could be demonstrated that 99Tc NMR spectroscopy allows similar investigations, although not all types of intermediates could be detected. The structure of the complex [Tc(CN-But)3(CO)3](NO3) was elucidated by X-ray diffraction analysis. Substitution with the protic ligand 2-mercaptoethanol HSCH2CH2OH revealed similar behaviour. Two intermediates were observed with IR spectroscopical methods. The final product was the dimer (NEt4)[Tc2(μ-SCH2CH2OH)3(CO)6], the structure of which was determined by X-ray diffraction.

Tumor Targeting of Mono-, Di-, and Tetravalent Anti-p185HER-2 Miniantibodies Multimerized by Self-associating Peptides

Multimerization of antibody fragments increases the valency and the molecular weight, both identified as key features in the design of the optimal targeting molecule. Here, we report the construction of mono-, di-, and tetrameric variants of the anti-tumor p185HER-2 single chain Fv fragment 4D5 by fusion of self-associating peptides to the carboxyl terminus. Dimeric miniantibodies with a synthetic helix-turn-helix domain and tetrameric ones with the multimerization domain of the human p53 protein were produced in functional form in the periplasm of Escherichia coli. We have directly compared these molecules and the single-chain Fv fragment in the targeting of SK-OV-3 xenografts. Tetramerization of the 4D5 antibody fragment resulted in increased serum persistence, significantly reduced off-rate, due to the avidity effect, both in surface plasmon resonance measurements on purified p185HER-2 and on SK-OV-3 cells. The99mtechnetium-tricarbonyl-labeled tetrameric 4D5-p53 miniantibody localized with the highest dose at the tumor and remained stably bound for at least 72 h. The highest total dose was 4.3% injected dose/g after 24 h, whereas the highest tumor-to-blood ratio was found to be 13.5:1 after 48 h, with a total dose of 3.2% injected dose/g. The tetramer shows no higher avidity than the dimer, presumably since the simultaneous binding to more than two antigen molecules on the surface of cells is not possible, and the improvement in performance over the dimer must at least be due in part to the molecular weight. These results demonstrate that multimerization by self-associating peptides can be used for the development of more effective targeting molecules for medical diagnostics and therapy.

Synthesis, 18F-Labeling, and in Vitro and in Vivo Studies of Bombesin Peptides Modified with Silicon-Based Building Blocks

The gastrin-releasing peptide receptor (GRPr) is overexpressed on various human tumors. The goal of our study was the synthesis of new 18F-labeled bombesin analogues for the PET imaging of GRPr expression in prostate tumor using a silicon-based one-step n. c. a. radiolabeling method. The silicon-containing building blocks were efficiently coupled to the N-terminus of the peptides via solid-phase synthesis. Radiolabeling of the obtained peptide precursors proceeded smoothly under acidic conditions (34-85% conversion). Using the di-tert-butyl silyl building block as labeling moiety, products containing a hydrolytically stable 18F-label were obtained. In in vitro receptor binding experiments 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH 2 ( 4b, IC50 = 22.9 nM) displayed a 12-fold higher binding affinity than 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-Gly-His(3Me)-Sta-Leu-NH2 ( 3b, IC50 = 276.6 nM), and 4b was therefore chosen for further evaluation. In vitro and ex vivo metabolite studies of [18F]4b showed no significant degradation. In biodistribution experiments, tumor uptake of [18F]4b was low and unspecific, whereas the GRPr-rich pancreas revealed a high and specific accumulation of the radiotracer. This study demonstrates the applicability of our silicon-based one-step n. c. a. radiolabeling method for the synthesis of new 18F-labeled bombesin derivatives. This innovative approach represents a general, straightforward access to radiolabeled peptides as PET imaging probes.

In vitro and in vivo evaluation of a 99mTc(I)-labeled bombesin analogue for imaging of gastrin releasing peptide receptor-positive tumors

A new radiolabeled bombesin analogue, [99mTc(I)-PADA-AVA]bombesin (7-14), was synthesized and in vitro and in vivo characterized. High affinity and rapid internalization were obtained in binding assays. A specific binding towards gastrin releasing peptide receptors-positive tissues, pancreas and tumor, was observed in CD-1 nu/nu mice bearing PC-3 prostate adenocarcinoma xenografts. We therefore conclude that [99mTc(I)-PADA-AVA]bombesin (7-14) might have promising characteristics for applications in nuclear medicine, namely for diagnosis of GRP receptor overexpressing tumors.

In vitro and in vivo evaluation of new radiolabeled neurotensin(8–13) analogues with high affinity for NT1 receptors

The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [99mTc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise.

Fluorine-18 click radiosynthesis and preclinical evaluation of a new 18F-labeled folic acid derivative

The folate receptor (FR) is highly expressed on most epithelial cancer cells, while normal cells show only restricted expression of FR. As a result, the FR is an ideal target for receptor-based molecular imaging and therapy of cancer and has become a promising target in oncology. To date, several folate-based chemotherapeutics and imaging probes such as radiopharmaceuticals for single photon emission computed tomography (SPECT) have been developed. However, an (18)F-labeled folic acid derivative suitable for positron emission tomography (PET) imaging that can be routinely applied is still lacking. In this study, a new fluorinated and radiofluorinated folic acid derivative, (18/19)F-click folate, was synthesized using click chemistry. In a convenient and very efficient two-step radiosynthesis, the isolated (18)F-click folate was obtained in good radiochemical yields of 25-35% with a specific activity of 160+/-70 GBq/micromol after <or=90 min synthesis time. The new compound was pharmacologically evaluated in vitro and in vivo. The affinity of the non-radioactive (19)F-click folate to the FR was determined in displacement studies with FR expressing KB tumor cells using (3)H-folic acid. In these in vitro binding studies, a nanomolar affinity with a K(i) of 9.76+/-3.13 nM was found for (19)F-click folate. The (18)F-labeled click folate derivative was then applied for in vivo PET studies and ex vivo biodistribution experiments using nude mice bearing KB tumor xenografts. The post mortem dissection experiments showed a high specific uptake of (18)F-click folate derivative in FR-expressing tissues. Uptake in KB tumor xenografts and kidneys (FR-positive tissue) amounted to 3.13%ID/g (94% specific blockade) and 16.53%ID/g (75% specific blockade), respectively. PET imaging using (18)F-click folate permitted a visualization of KB tumors, and blockade studies confirmed the specific accumulation of the radiotracer in vivo. However, strong hepatobiliary excretion of the new tracer led to elevated accumulation of radioactivity in the abdominal region. In conclusion, the click chemistry approach is convenient to accomplish and provided high radiochemical yields of (18)F-click folate. The new tracer showed good in vitro but limited in vivo properties. Ultimately, the (18)F-click folate emphasizes the potential of (18)F-labeled folates for receptor-based tumor PET imaging.