Carmen Wängler

Antibody-dendrimer conjugates: the number, not the size of the dendrimers, determines the immunoreactivity.

Radioimmunotherapy using antibodies with favorable tumor targeting properties and high binding affinity is increasingly applied in cancer therapy. The potential of this valuable cancer treatment modality could be further improved by increasing the specific activity of the labeled proteins. This can be done either by coupling a large number of chelators which leads to a decreased immunoreactivity or by conjugating a small number of multimeric chelators. In order to systematically investigate the influence of conjugations on immunoreactivity with respect to size and number of the conjugates, the anti-EGFR antibody hMAb425 was reacted with PAMAM dendrimers of different size containing up to 128 chelating agents per conjugation site. An improved dendrimer synthesis protocol was established to obtain compounds of high homogeneity suitable for the formation of defined protein conjugates. The quantitative derivatization of the PAMAM dendrimers with DOTA moieties and the characterization of the products by isotopic dilution titration using (111)In/(nat)In are shown. The DOTA-containing dendrimers were conjugated with high efficiency to hMAb425 by applying Sulfo-SMCC as cross-linking agent and a 10- to 25-fold excess of the thiol-containing dendrimers. The determination of the immunoreactivities of the antibody-dendrimer conjugates by FACS analysis revealed a median retained immunoreactivity of 62.3% for 1.7 derivatization sites per antibody molecule, 55.4% for 2.8, 27.9% for 5.3, and 17.1% for 10.0 derivatization sites per antibody but no significant differences in immunoreactivity for different dendrimer sizes. These results show that the dendrimer size does not influence the immunoreactivity of the derivatized antibody significantly over a wide molecular weight range, whereas the number of derivatization sites has a crucial effect.

Multimerization of cRGD peptides by click chemistry: synthetic strategies, chemical limitations, and influence on biological properties.

Integrin ανβ3 is overexpressed on endothelial cells of growing vessels as well as on several tumor types, and so integrin‐binding radiolabeled cyclic RGD pentapeptides have attracted increasing interest for in vivo imaging of ανβ3 integrin expression by positron emission tomography (PET). Of the cRGD derivatives available for imaging applications, systems comprising multiple cRGD moieties have recently been shown to exhibit highly favorable properties in relation to monomers. To assess the synthetic limits of the cRGD‐multimerization approach and thus the maximum multimer size achievable by using different efficient conjugation reactions, we prepared a variety of multimers that were further investigated in vitro with regard to their avidities to integrin ανβ3. The synthesized peptide multimers containing increasing numbers of cRGD moieties on PAMAM dendrimer scaffolds were prepared by different click chemistry coupling strategies. A cRGD hexadecimer was the largest construct that could be synthesized under optimized reaction conditions, thus identifying the current synthetic limitations for cRGD multimerization. The obtained multimeric systems were conjugated to a new DOTA‐based chelator developed for the derivatization of sterically demanding structures and successfully labeled with 68Ga for a potential in vivo application. The evaluated multimers showed very high avidities—increasing with the number of cRGD moieties—in in vitro studies on immobilized ανβ3 integrin and U87MG cells, of up to 131‐ and 124‐fold, respectively, relative to the underivatized monomer.

Kit-Like 18F-Labeling of Proteins: Synthesis of 4-(Di-tert-butyl[18F]fluorosilyl)benzenethiol (Si[18F]FA-SH) Labeled Rat Serum Albumin for Blood Pool Imaging with PET

Radiosyntheses of 18F-radiopharmaceuticals for positron emission tomography (PET) normally require an extraordinarily high effort of technical equipment and specially trained personnel. We recently reported a novel method for the introduction of fluorine-18 into peptides for PET-imaging based on silicon-18F-chemistry (SiFA technique). We herewith introduce the first SiFA-based Kit-like radio-fluorination of a protein (rat serum albumin,RSA) and demonstrate its usefulness for in vivo imaging with microPET in normal rats as well as in a rat heterotropic transplanted heart model. As a labeling agent, we prepared 4-(di-tert-butyl[18F]fluorosilyl)benzenethiol (Si[18F]FASH)by simple isotopic exchange in 40-60% radiochemical yield (RCY) and coupled it directly to a Sulfo-SMCC derivatized RSA in an overall RCY of 12% within 20-30 min. The technically simple labeling procedure does not require any elaborated purification procedures and is a straightforward example of a successful application of Si-18F chemistry for in vivo imaging with PET.

One-step 18 F-labeling of peptides for positron emission tomography imaging using the SiFA methodology

Here we present a procedure to label peptides with the positron-emitting radioisotope fluorine-18 (18F) using the silicon-fluoride acceptor (SiFA) labeling methodology. Positron emission tomography (PET) has gained high importance in noninvasive imaging of various diseases over the past decades, and thus new specific imaging probes for PET imaging, especially those labeled with 18F, because of the advantageous properties of this nuclide, are highly sought after. N-terminally SiFA–modified peptides can be labeled with 18F− in one step at room temperature (20–25 °C) or below without forming side products, thereby producing satisfactory radiochemical yields of 46 ± 1.5% (n = 10). The degree of chemoselectivity of the 18F-introduction, which is based on simple isotopic exchange, allows for a facile cartridge-based purification fully devoid of HPLC implementation, thereby yielding peptides with specific activities between 44.4 and 62.9 GBq μmol−1 (1,200–1,700 Ci mmol−1) within 25 min.

One-Step 18F-Labeling of Carbohydrate-Conjugated Octreotate-Derivatives Containing a Silicon-Fluoride-Acceptor (SiFA): In Vitro and in Vivo Evaluation as Tumor Imaging Agents for Positron Emission Tomography (PET)

The synthesis, radiolabeling, and initial evaluation of new silicon-fluoride acceptor (SiFA) derivatized octreotate derivatives is reported. So far, the main drawback of the SiFA technology for the synthesis of PET-radiotracers is the high lipophilicity of the resulting radiopharmaceutical. Consequently, we synthesized new SiFA-octreotate analogues derivatized with Fmoc-NH-PEG-COOH, Fmoc-Asn(Ac₃AcNH-β-Glc)-OH, and SiFA-aldehyde (SIFA-A). The substances could be labeled in high yields (38 ± 4%) and specific activities between 29 and 56 GBq/μmol in short synthesis times of less than 30 min (e.o.b.). The in vitro evaluation of the synthesized conjugates displayed a sst2 receptor affinity (IC₅₀ = 3.3 ± 0.3 nM) comparable to that of somatostatin-28. As a measure of lipophilicity of the conjugates, the log P(ow) was determined and found to be 0.96 for SiFA-Asn(AcNH-β-Glc)-PEG-Tyr³-octreotate and 1.23 for SiFA-Asn(AcNH-β-Glc)-Tyr³-octreotate, which is considerably lower than for SiFA-Tyr³-octreotate (log P(ow) = 1.59). The initial in vivo evaluation of [¹⁸F]SiFA-Asn(AcNH-β-Glc)-PEG-Tyr³-octreotate revealed a significant uptake of radiotracer in the tumor tissue of AR42J tumor-bearing nude mice of 7.7% ID/g tissue weight. These results show that the high lipophilicity of the SiFA moiety can be compensated by applying hydrophilic moieties. Using this approach, a tumor-affine SiFA-containing peptide could successfully be used for receptor imaging for the first time in this proof of concept study.

A Universally Applicable 68Ga-Labeling Technique for Proteins

Although protein-based PET imaging agents are projected to become important tracer molecules in the future, the labeling of complex biomolecules with PET radionuclides is inexpedient and, most of the time, challenging. Methods: Here we present a straightforward labeling chemistry to attach the versatile radionuclide 68Ga to proteins. Introducing the 68Ga chelating agent NODA-GA-T (2,2′-(7-(1-carboxy-4-(2-mercaptoethylamino)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid) by reaction with proteins chemically processed with sulfo-SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt) results in labeling precursors, enabling a simple and rapid kit-labeling procedure that requires no workup of the radiolabeled proteins. Various 68Ga- proteins were labeled using this method, and the radiochemical yields and specific activities of the labeled proteins were determined. To show that the radiotracers are applicable for in vivo studies, proof-of-concept small-animal PET images were acquired in healthy rats using 68Ga rat serum albumin for blood-pool imaging and 68Ga-annexin V for apoptosis imaging in mice with a left ventricular myocardial infarction. Results: The proteins could be modified, yielding 1.2–1.7 68Ga-labeling sites per protein molecule. All investigated proteins could be labeled in high radiochemical yields of 95% or more in less than 10 min in 1 step, using acetate-buffered medium (pH 3.5–4.0) at room temperature without any further purification. The labeled proteins displayed specific activities of 20–45 GBq/μmol (540–1,200 Ci/mmol). In the proof-of-concept in vivo studies, 68Ga rat serum albumin and 68Ga-annexin V were successfully used for in vivo imaging. Both radiotracers showed a favorable biodistribution in the animal models, thus demonstrating the usefulness of the developed approach for the kit 68Ga labeling of proteins. Conclusion: The preprocessing of proteins proceeds in high chemical yields and with high protein recovery rates after purification. These precursors can be stored for several months at −20°C without degradation, and 68Ga labeling can be performed in a 1-step kit-labeling reaction in high radiochemical yields. Two of the derivatized model proteins were successfully used in proof-of-concept in vivo imaging studies to prove the applicability of this kit 68Ga-labeling technique.

(89)Zr, a radiometal nuclide with high potential for molecular imaging with PET: chemistry, applications and remaining challenges.

Molecular imaging—and especially Positron Emission Tomography (PET)—is of increasing importance for the diagnosis of various diseases and thus is experiencing increasing dissemination. Consequently, there is a growing demand for appropriate PET tracers which allow for a specific accumulation in the target structure as well as its visualization and exhibit decay characteristics matching their in vivo pharmacokinetics. To meet this demand, the development of new targeting vectors as well as the use of uncommon radionuclides becomes increasingly important. Uncommon nuclides in this regard enable the utilization of various selectively accumulating bioactive molecules such as peptides, antibodies, their fragments, other proteins and artificial structures for PET imaging in personalized medicine. Among these radionuclides, 89Zr (t1/2 = 3.27 days and mean Eβ+ = 0.389 MeV) has attracted increasing attention within the last years due to its favorably long half-life, which enables imaging at late time-points, being especially favorable in case of slowly-accumulating targeting vectors. This review outlines the recent developments in the field of 89Zr-labeled bioactive molecules, their potential and application in PET imaging and beyond, as well as remaining challenges.

Oxalic Acid Supported Si–18F-Radiofluorination: One-Step Radiosynthesis of N-Succinimidyl 3-(Di-tert-butyl[18F]fluorosilyl)benzoate ([18F]SiFB) for Protein Labeling

N-Succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB), a novel synthon for one-step labeling of proteins, was synthesized via a simple (18)F-(19)F isotopic exchange. A new labeling technique that circumvents the cleavage of the highly reactive active ester moiety under regular basic (18)F-labeling conditions was established. In order to synthesize high radioactivity amounts of [(18)F]SiFB, it was crucial to partially neutralize the potassium oxalate/hydroxide that was used to elute (18)F(-) from the QMA cartridge with oxalic acid to prevent decomposition of the active ester moiety. Purification of [(18)F]SiFB was performed by simple solid-phase extraction, which avoided time-consuming HPLC and yielded high specific activities of at least 525 Ci/mmol and radiochemical yields of 40-56%. In addition to conventional azeotropic drying of (18)F(-) in the presence of [K(+)⊂2.2.2.]C(2)O(4), a strong anion-exchange (SAX) cartridge was used to prepare anhydrous (18)F(-) for nucleophilic radio-fluorination omitting the vacuum assisted drying of (18)F(-). Using a lyophilized mixture of [K(+)⊂2.2.2.]OH resolubilized in acetonitrile, the (18)F(-) was eluted from the SAX cartridge and used directly for the [(18)F]SiFB synthesis. [(18)F]SiFB was applied to the labeling of various proteins in likeness to the most commonly used labeling synthon in protein labeling, N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). Rat serum albumin (RSA), apo-transferrin, a β-cell-specific single chain antibody, and erythropoietin were successfully labeled with [(18)F]SiFB in good radiochemical yields between 19% and 36%. [(18)F]SiFB- and [(18)F]SFB-derivatized RSA were directly compared as blood pool imaging agents in healthy rats using small animal positron emission tomography. Both compounds demonstrated identical biodistributions in healthy rats, accurately visualizing the blood pool with PET.

PAMAM structure-based multifunctional fluorescent conjugates for improved fluorescent labelling of biomacromolecules

Fluorescent probes are of increasing interest in medicinal and biological applications for the elucidation of the structures and functions of healthy as well as tumour cells. The quality of these investigations is determined by the intensity of the fluorescence signal. High dye/carrier ratios give strong signals. However, these are achieved by the occupation of a high number of derivatisation sites and therefore are accompanied by strong structural alterations of the carrier. Hence, polyvalent substances containing a high number of fluorescent dyes would be favourable because they would allow the introduction of many dyes at one position of the compound to be labelled.A large number of different dyes have been investigated to determine the efficiency of coupling to a dendrimer scaffold and the fluorescence properties of the oligomeric dyes, but compounds that fulfil the requirements of both strong fluorescence signals and reactivities are rare. Herein we describe the synthesis and characterisation of dye oligomers containing dansyl-, 7-nitro-2,1,3-benzoxadiazol-4-yl- (NBD), coumarin-343, 5(6)-carboxyfluorescein and sulforhodamine B2 moieties based on polyamidoamine (PAMAM) dendrimers. The PAMAM dendrimers were synthesised by an improved protocol that yielded highly homogeneous scaffolds with up to 128 conjugation sites. When comparing the fluorescent properties of the dye oligomers it was found that only the dansylated dendrimers met the requirements of enhanced fluorescence signals. The dendrimer containing 16 fluorescent dyes was conjugated to the anti-epidermal-growth-factor receptor (EGFR) antibody hMAb425 as a model compound to show the applicability of the dye multimer compounds. This conjugate revealed a preserved immunoreactivity of 54%.We demonstrate the applicability of the dye oligomers to the efficient and applicable labelling of proteins and other large molecules that enables high dye concentrations and therefore high contrasts in fluorescence applications.

Improved work-up procedure for the production of [18F] flumazenil and first results of its use with a high-resolution research tomograph in human stroke

INTRODUCTION The central benzodiazepine receptor (cBZR)-gamma-aminobutyric acid (GABA(A)) receptor complex in the human brain plays an important role in many neurological and psychiatric disorders. (18)F-Labeled flumazenil ([(18)F]FZ) provides a potentially useful tracer to investigate those disorders by means of positron emission tomography (PET). METHODS [(18)F]Flumazenil was synthesized from its nitro-precursor Ro 15-2344 in DMF at high temperatures between 150 degrees C and 160 degrees C. Other solvents like acetonitrile and dimethylsulfoxide were also investigated as reaction media. A new HPLC method for the final purification of [(18)F]FZ was developed to circumvent some difficulties associated with a previously published procedure sometimes led to a contamination of [(18)F]FZ with Ro 15-2344. The final purification of the radiotracer was achieved using a Waters Symmetry Prep C18 HPLC column with elution with 0.05 M sodium acetate (NaOAc) buffer (pH 5)/THF/MeOH (80:10:10). RESULTS [(18)F]FZ could be synthesized in reproducible radiochemical yields (RCYs) of 15-20% (decay corrected to EOB) after 80 min overall synthesis time. The synthesized [(18)F]FZ was applied for the first time in a human PET study in a patient with ischemic right middle cerebral artery stroke using the HRRT high-resolution research scanner (Siemens Medical Solution, Knoxville, TN, USA). CONCLUSIONS [(18)F]FZ is a potentially useful GABA receptor-binding PET ligand. A modified procedure for its preparation in reproducibly high radiochemical yields has been described and the [(18)F]FZ thus produced has been used successfully in a pilot clinical study.